Per C6 Abortion Cell Lines

UNITED STATES OF AMERICA

..

FOOD AND DRUG ADMINISTRATION CENTER FOR BIOLOGICS EVALUATION AND RESEARCH + + + + +

VACCINES AND RELATED BIOLOGICAL PRODUCTSADVISORY COMMITTEE + + + + +

MEETING

WEDNESDAY, MAY

16,

2001

Inn

Gaithersburg,

This t1 or car: from t semia Drug L P8pres

Ballroom, Village Dr.

Holiday Avenue,

Robert

Gaithersburg,

2

Maryland,

S. Daum, Acting

Chair,

Montgomery

at 9:00 a.m.,

presiding.

PRESENT: ROBERT S. DAUM, M.D.,

Acting

Chair

C. ESTUARDO AGUILAR-CORDOVA, M.D., DONALD BLAIR,

Ph.D.

Ph.D.

JOHN COFFIN, Ph.D. JAMES COOK, M.D. MICHAEL DECKER, M.D. PAMELA S. DIAZ,

M.D.,

Member

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2 .PRESENT (Continued)

i

ALEX J. VANDER EB, Ph.D. WALTER L. FAGGETT, M.D.,

Member

BARbARA LOE FISHER, MemberJUDITH D. GOLDBERG,.Sc.D., DIANE E. GRIFFIN,

M.D.,

member

Ph.D.,

Member

STEPHEN HUGHES, Ph.D. SAMUEL L. KATZ, M.D., KWANGSIK KIM, M.D., STEVE KOHL, M.MD.,

Member Member

Member

,PAMELA McINNES, D.D.S., PHILIP

Msc.(Dent.)

MINOR, Ph.D.

LAWRENCEMOULTON, Ph.D. MARTIN MYERS, M.D. SUZETTE PRIOLA, Ph.D. DAVID S. STEPHENS, M.D.,

Member

SIDNEY WOLFE, M.D. NANCY CHERRY, Executive

Secretary

NEAL R. GROSS (202) 234433

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C-O-N-T-E-N-T-S

Conflict

of

Introductions

Interest

Statement

. . . . . . . . .

4

. . . . . . . . . . . . . . . . .

4

Introduction to the Session on Designer Cell Substrate, Dr.AndrewLewis . . . . ...16 Designer Cell Substrates for Vaccine Development: Concepts and Issues, Dr. Steve Hughes . . . . . . . . . . . . . 35 Adenovirus Biology as Related to Development and Use of Adenovirus Vectors, Dr. Estuardo Aguilar-Cordova . . . . . . . . . 54 Adenovirus Transformation of Human Cells and.the Development of 293 and PER.CG for the Manufacture of Defective Adenovirus Vectors, Dr.AlexvanderEb . . . . . . . . . . . 77 Adenovirus Transformed Cell Transformed Cell-Host Determine their Tumor Dr. James Cook . . . Quantitative Assessment Residual DNA, Dr.

Tumorigenicity and Interaction that Forming Capability, . . . . . . . . . .

of the Risks Keith Peden

100

of . . . . .

131

Introduction to Adventitious Agent Issues, Dr. Philip Krause, . . . . . . . . . . .

161

Transmissible Spongiform Encephalopathy Agents as an Issue in the Use of Neoplastic Cell Substrates, Dr. Sue Priola . . . . . . .

168

- Adventitious Agent Testing of Neoplastic Cell Substrates, Dr. Philip Krause . . . . .

196

Review

of OVRR-CBER Issues with the Use of Adenovirus-vectored Vaccines and their Complementing Designer Cell Substrates, Dr. Hana Golding . . . . . . . . . . . .

Committee

Discussion

. . . . . . . . . . . . .

228 239

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4

1

P-R-O-C-E-E-D-I-N-G-S

2

(9:Ol

.3

ACTING

4

will

begin

5

Nancy Cherry,

6

statement.

CHAIRMAN DAUM:

our session who will

8

welcome you all

9

the

turning

read

MS. CHERRY:

7

to this

Good morning.

We

the floor

to

the.conflict

First

of

meeting,

over

of interest

all,

I'd

and then

like

I will

to read

statement. The

10

following

11

conflict

of

interest

12

Session

2 of

the

13

Products

Advisory

announcement

issues Vaccines

Committee

15

on adventitious

16

and issues

17

novel

18

manufacture

related

and

viral

appointed

for

22

existed,

23

all

24

participants.

25

following

cell

on discussion

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substrates

this

voting

DNA of used

to

if

interests As

disclosures

members

have

been

session. any conflicts

the agency reviewed

financial

2001.

vaccines.

To determine

21

Biological

tumorigenicitytesting,

cell

No temporary

19

this

on May 16th,

is focused

to residual

neoplastic

with

Related

meeting

agent testing,

addresses

associated and

This open session

14

20

with

a.m.1

a

the submitted .reported

result are

of being

by this

of interest agenda and the

meeting

review,

the

made regarding

the

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5

1

discussion

May 16th. Griffin,

2

Drs.

3

each been granted

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the discussions.

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Drs. Blair,

Drs.

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12

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associations

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of Dr.

In

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and Moulton

Cook,

firms

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fee

and be or

discussions.

18 USC 208 and with

has

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Kim,

could

Committee

of

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none of

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which

McInnes,

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21

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Griffin,

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Hughes,

sufficient

18 USC

determinations

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Goldberg,

Stephens,

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reported

scientific

a

or for

that

the

guest

are

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he

advice

on

line.

addition,

the

agency

has

determined

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6 1

that

the

services

of

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voting

guest

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is

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President

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reported

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industry

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are also

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Medical

and interest

addition, with

9

major

manufacturers.

specific

products

12

which

13

the participantsare

14

themselves

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be noted

FDA's

16

that

or firms

participants

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the public

Their

respect

to

you state

your name and affiliation

19

previous

financial.involvement

20

products

you wish

24

Information

all

involve

interest,

recusals

other

will

meeting

of fairness and-any

with

of

all

addressed by written

waivers in

request

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firm

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and

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under

the

Freedom

are of

Act. And I do have one other

25

with

to comment on.

Copies

available

and

record.

18

23

has

on the agenda and for

we ask in the interest

determinations

be

of the need to exclude

participants,

22

could

employer

have a financial

17

21

He

the discussions

reminded

With

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from the discussions. for

Vice

Affairs.

Decker's

university

In the event

11

as the

in a firm

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associations

10

Pasteur

Dr.

discussion.

8

vaccine

as a non-

essential.

Scientific

committee

In

Decker

announcement.

The

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7 1

Committee

management specialists

2

to

3

sitting

4

being

5

have any problems,

put

this

assisted

today

by Rosanna

I

guess,

Denise

both

Royster

Harvey,

is

and if

you

see them. Thank

you

very

much, Nancy. There's the

room

that is

a peculiar

seems to

10

anyone

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whispering,

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and it's

my echo going

13

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was speaking

speaking.

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We had it

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around

sounds

after

Can you give sitting

microphone

be resonating

and I realize

14 15

are,

desk now.

please

did so muchwork

ACTING CHAIRMAN DAUM:

8 9

together

out at the front

6 7

meeting

that

it's

me

when Dr.

also.

a thought?

Maybe I'm just

here.

16

PARTICIPANT:

17

ACTING CHAIRMAN DAUM: When I speak I am.

18

Also,

cell

phones,

19

you can't

use on airplanes,

20

either.

Different

21

tone

22

deliberations,

23

everybody

now thought

24

or

phone

25

Committee.

of

cell

the

Are you hearing

beepers,

please

all

don't

They really

discussion

and

the

much

be

that

I'd

very

about whether could

ring

now?

the things

use them here

reason.

and

it

distract

the

Committee grateful

if

they have a beeper and

disrupt

the,

NEAL R. GROSS (202) 2344433

COURT REPORTERS AND TRANSCRIBERS 1323 RHODE ISLAND AVE., N.W. WASHINGTON, D.C. 20005-3701

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1

I would

2

around

the table

3

this

4

slight

5

we usually

6

Griffin

7

the standard

8

ask

9

working

morning,

and I would

discrimination

and that

Committee

everybody

else,

that

affiliation

12

two.

Why are they

13

general

15

orienting

16

Griffin,

everyone would

only

consulting

I'm

which

are

Myers

and

say who they

are

Dr.

in

of explain

how

one sentence

or

for

in

in terms

of

issue.

the

discussion.

us off,

So,

Dr.

please?

So I am Diane the

Dr.

to

would be helpful

toward

with

to our Committee

particular that

the way

I'm going

but sort

them here

be a

chair

of

Griffin the

from

Molecular

Microbiology and Immunology Department in the School __ '.i of Public Health, and I'm going to explain a little ~-bit

about

myself. I'm

22 23

as Ms. Fisher,

is,

you start

Hopkins.

start

with

DR. GRIFFIN:

17

21

this

.we'll

to not

there

unless

starting

gets

I think

Johns

is

to go

themselves

to ask that

and then

affiliation

or for

introduce

members,

our way around,

11

20

like

and come down as far

14

a few minutes

in the process,

do it,

and what their

19

to take

and have people

10.

18

like

viral

interested

in

the

pathogenesis

of

infections.

24

ACTING cwmm~~

DAD-M: Perfect.

25

DR. STEPHENS:

I'm

David

Stephens

from

NEAL R. GROSS (202) 234-4433

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9 University,

1

Emory

2

Infectious

3

virologist.

Diseases.

the

need to be less

pass to the next

explicit

Division

a bacteriologist,

ACTING CHAIRMAN DAUM: in this

of not

a

person.

Committee

members

regard.

(Laughter.)

6

ACTING CHAIRMAN DAUM: This

7

but rather

8

expose,

9

understand

10

of

I'm

So 1'11

4 5

Director

fact,

an opportunity

for

why the consultants

that

is not a total

the Committee

to

are here today,

in

are.

11

Dr.

12

DR. GOLDBERG: Hi.

13

the Director

14

School

Goldberg.

of Biostatistics,at

DR. infectious

17

of his

KATZ :

disease career

New York University,

I'm

person

studying

19

infectious

disease

20

Infectious

Diseases

21

Health. DR.

22 23

infectious

24

University,

with

Pamela

spent

Diaz,

person

and

for

the

Chicago

I'm

Steve

the

most

pediatric

Director

of

Department

of

Kohl,

and at the Argonne

an expertise

DR. KIM:

a pediatric

from Duke who's

I'm

KOHL:

diseases

Sam Katz,

vaccines.

DR. DIAZ:

18

25

I'm

of Medicine.

15 16

Judy Goldberg.

pediatric

Health

in viral

I'm Kwang Sik Kim.

Science

immunology. I'm head of

NEAL R. GROSS (202) 2344433

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10 1

-pediatric

infectious

2

My work

3

infectious

4

in 'pediatrics.

diseases

has been

primarily

diseases,

the

6

of

7

nonprofit

8

safety.

National

Barbara.Loe

Vaccine

organization

10

Director

of

11

Background:

12

interested

13

models

14

Pediatrics.

the

I'm

Martin

National

in

16

Director,

Division

17

Diseases,

National

18

Diseases.

NIAID

19

through

20

and clinical

public

of

money, research

I'm

22

emeritus

professor

23

expertise

in

24

general.

I'm

25

advisor

to

viral still Crucell,

about

Office.

animal Chairman

Pamela McInnes, and

of Allergy

on basic,

funder applied,

diseases.

I am Alex

at the University

van der

of Leiden,

transformation

member

Deputy

and Infectious

expenditure

a

of

Infectious

an important

in the

the

diseases

of course,

active

vaccine

Program

former

in infectious

a

I'm

Microbiology

DR. VAN DER EB:

21

Center,

particularly

Institute is,

President

infectious

infections;

MS. McINNES:

15

of

Myers.

Vaccine

pathophysiology,

of Herpes,viral

Fisher,

concerned

pediatrician in

pathogenesis

Information

that's

DR. MYERS:

9

on the

School.

primarilyonbacterialinfections

MS. FISHER:

5

at Johns Hopkins

and lab of.

Eb, with

cancer

in

and scientific the

Scientific

NEAL R. GROSS (202) 234-4433

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11

1

..

Advisory

Committee. DR. DECKER: I'm Dr. Michael

2 3

a member of the Departments

4

Infectious

5

for,

6

in, clinical

7

joined

8

Scientific

9

through

10

industry

Diseases

oh,

at Vanderbilt

ten or 15 years research

Aventis,

Pasteur

a typical

DR. Aguilar.

13

Initiative,

14

because

15

in gene therapy

as

federal

I'm

Vice

process,

18

Microbiology

19 .20

Director of the -also part-time

21

guess,

because

22

years

has

23

retroviruses

24

issues

the

I've

President

for

here because

AGUILAR-CORDOVA:

I'm

Estuardo

with

Gene

to VerPAC.

the

Harvard

Therapy

been asked to come here primarily vectors

and their

use

applications. John Coffin.

Department

related

involved

vaccine

DR. COFFIN: in

where

Recently

and I'm

of my work in antiviral

17

and

I am the

and I've

16

Medicine

been actually

Affairs,

representative

12

I've

I'm

University,

and vaccines.

and Medical

11

of

at Tufts NCI's

work

Biology

and also

HIV Drug Resistance grower.

my research

been

I'm a professor

Molecular

University

cranberry

engaged

over in

and how they

part-time Program and

And I'm quite

and

here,

I

a number

of

understanding transform

how

cells

and

to that. DR. COOK:

25

of Preventive

Decker.

I'm

Jim

Cook.

I’m

Chief

of

NEAL R. GROSS (202) 234-4433

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12 1

> Infectious

Disease

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my

3

expression,

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interest

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Mechanism

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spend

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safety

and vaccine

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14

Department

15

Hopkins

University

16

Health,

and I'm

of

of

Center

Control

in the United

20

quality

control

21

viral

22

contamination,

I've

and

I

working

Philip

the of

Minor.

We're

the Johns

Public

issues get

I'm

from

Standards

and

concerned

with

and regulation

of

involved

of biological

in

viral

products.

I'm Sid Wolfe.

training,

from

School

Biological

Kingdom.

spent

on vaccine

geneticist.

and we also

by clinical

University,

at

I'm

DR. WOLFE:

of

a

Microbiology

of

issues

history

I'm

Ketner

and quality

vaccines,

for

Moulton.

Gary

an adenovirus

Institute

of

andtumorigenesis.

now Bloomberg

19

ago,

the

Chief

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Molecular

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30 years

in host..

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efficacy

18

25

response

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DR. MINOR:

internist

the

Hopkins

DR. KETNER:

24

affects

activity

Johns

majority

23

and how it

Don.Blair.

MOULTON:

10

National

gene

at the NC1 and have a long

DR.

17

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in DNAbiological

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adenoviral

to the inflammatory

DR. BLAIR:

9

and

is

especially

response

of Illinois,

and since

most of my time

I'm a general leaving

NIH

at the Public

NEAL R. GROSS (202) 234-4433

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13

1

-.Citizens

Health

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relate

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Research FDA, drugs,

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infection,

15

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16

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that's

I'm in

an

30 years

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to

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tissue

culture

DR. HUGHES:

I'm

asked

an off,

about cells

Steve

most

to

from the Rocky

Institutes

information

the

come at least

I'm Sue Priola

Laboratories,

and

sometimes

the FDA for

issues

and

11

with

that

and I think

closely,

certainly

attention,

activities

problems.

and important

10

in

biologics,

way, but closely

and sort

7

Group

off,

off

of Health,

and

infectivity

TSE

and

the

Hughes.

risks

I'm

from

17

the HIV Drug Resistance

Program of the NCI, and I have

18

a longstanding

in retroviruses.and

19

vectors.

20 21 22

-..G_ Daum.

ACTING CHAIRMAN DAUM: I'm

from

--

I'm

with

retroviral

And

I'm

Robert

parainfluenza

virus

infection. (Laughter.)

23

ACTING

24 25

interest

University

of

CHAIRMAN DAUM:

Chicago.

I'm

head of

~'rn the

from

the

Section

of

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14 1

.PediatriC

Infectious

2

include

antimicrobially

3

positive

bacteria,

4

closet

5

vaccines

6

rates

in inner

concerns

city

members and guests,

9

a very help

these

from the FDA, who will

14

so-called

16

podium,

17

and just

could

20

Vaccines

21

vaccines,

22

on HIV.

cell Dr.

I'd

25

in viral

like

to

to move on with

on Dr.

is tell

walking

Andrew Lewis session

up

us who they

of brief,

on

Products

involved

to

the

are also

USA Todav format?

Yes, my name is Keith

of Viral

I'm

today

substrates.

and as a nighttime

of DNA Viruses.

We have obviously

us to this

Lewis

at CBER. We're

24

of

issues.

DR. KRAUSE: Phil

23

my

everybody,

consultants

introduce

in the same kind

I'm in the Division

of

and call

the FDA folks

19

and

immunization

I welcome

point

DR. PEDEN:

18

that,

important

13

While

improving

panel

the body of the meeting

15

job,

Gram

evaluation

to our meeting.

12

designer

in

children.

And at this

11

stress

my day

for

distinguished

us with

My interests

clinical

strategies

8

10

that's

And so with

7

there.

induced and

research and

Diseases

Peden.

in the Office

of

in the regulation

of

job we do some research

Krause in the Laboratory

interested

in viral

latency

and

detection. NEAL R. GROSS

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15

DR. GOLDING:

1 2

Chief

of

the

3

Division

4

regulation

5

has been

6

development..

focused

in

entry

I

DR. STEPHENS:

world

and HIV vaccine

Thank

you

very

am Diane

Griffin

from

I'm

David

Stephens

from

Emory University. DR. GOLDBERG: Judy Goldberg

from New York

University. Sam Katz from Duke University. Pamela Diaz,

Chicago Department

of Health. DR. KOHL:

18

Science

School

Vaccine

Kohl,

Kwang Sik

Information

Barbara

Program

Health

Kim,

Johns

Hopkins

Loe Fisher,

National

Center:

DR. MYERS: MartinMyers,

24

Argonne

of Medicine. MS. FISHER:

22

Steve

University. DR. KIM:

20

25

much involved

Johns Hopkins.

DR. DIAZ:

23

in

and my scientific

DR. GRIFFIN:

16

21

I'm very

on HIV cell

DR. KATZ:

19

Research

Retrovirus

Product.

15

17

the

kindly.

13 14

I'm

ACTING CHAIRMAN DAUM:

11 12

of

of HIV vaccine,

9 10

Laboratory

of Viral

7 8

I'm Hana Golding.

National

Vaccine

Office. NEAL R. GROSS

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16 DR. COFFIN:

1

2

and sometimes

4

COOK:

Jim

DR. BLAIR:

6

DR. MOULTON: Larry

Don Blair,

DR. KETNER:

University

Institute

Johns Hopkins

Gary Ketner,

of Biological

Health

Research

Minor

Standards

DR. WOLFE:

11

Sid

Johns Hopkins. from the National

in the U.K.

Wolfe,

Public

Citizens

Group.

13

DR. HUGHES: Steve

14

ACTING CHAIRMANDAUM: And I'mRobert

15

of

NCI.

Moulton,

DR. MINOR: Philip

9

12

Cook,

University.

18

10

University

Illinois.

5

7

Tufts

NCI. DR.

3

John Coffin,

from

the University

16

I'm Andrew Lewis,

18

need to

19

this

cut

better

20

the

Daum

as it lights

And by way of says on this

introduction,

slide.

down a bit.

Maybe we

Can people

see

now? I'm

the

21

Viruses,

22

FDA about

a little

23

basically

a 30-year

24

of

25

transformed

Health

NCI.

of Chicago.

DR. LEWIS:

17

Hughes,

Division

Chief

of

of Viral

studying

over

Laboratory

Products.

five

career

the

years

of

DNA

I came to the ago,

at the National

adenoviruses

and

having

spent

Institutes adenovirus

cells. NEAL R. GROSS

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17

1

My role

2

twofold.

3

Office

4

cell

5

second,

6

substrates

7

for

The first

substrates to

to

for

viral

the

vaccine

status

of

development

topic

issues

session

is the

to the use of neoplastic

the

and the

today's

review

approach

introduce

vaccine

of

and,

designer

associated

with

cell

their

use

manufacture. Is

about

is

of Vaccines'

8 9

in introducing

focusing

this

better?

this

10

Okay.

11

Several

slide?

of the topics

have evolved

from studies

13

vitro

culture

14

development

To evolved

from these

17

the speakers

today,

18

mean when

we say

19

transformation,

20

oncogenicity.

that

I've

defined

will

in this

tumorigenicity

23

spontaneously

transformed

24

cells

types

is

may be either

its

is,

broadest cells,

that's

slide

what we

cells,

for

cell

and viral

our

sense virus

of immortalized

tumorigenic

in

be used by some of

line

today,

or other

terminology

neoplastic

22

using

of neoplastic

we need

cells

today

models.

the

fields

Neoplastic in

oncology,

using) animal

cell

used

see

better?

in studies

understand

16

21

you

for discussion

of viral

systems

in vivo

15

Is that

could

Thank you.

12

tissue

Keith,

cell

discussion to

include

transformed lines

that

or non-tumorigenic.

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18 1

Transformation

2

normal

3

oncogenes

4

neoplktic

cells

are

or

changed

spontaneous

by

viral

events

Tumorigenicity

6

neoplastic

7

and develop

8

and oncogenicity

9

cellular

10

animal

cells

by or

to

11 12

vaccine

13

A number

into

which

cellular

become

immortal

14

reconsider

15

development,

16

discussion

convert cells.

Now,

the

manufacture

the growth

19

delivery

factors

are

today.are

of

contributing

cells

cell

since to the

that

1954.

vaccine

are related

lines

capable

viral

vectors

for

need to

for

in this

or

an injected

substrates

factors

animals,

or viral

neoplastic

presented

of defective

20

of

cell

and those

systems

into

cells

of

to multiply

has been discouraged

First,

18

culture

of a virus

the

use

neoplastic

17

ability

when injected

is the ability to

the

in tissue

tumors

tumor

of

if.

growing

genes into

to the

slide.

of complimenting used as antigen

and hence of vaccines.

Second vectored

process

a

cells.

5

21

is

is

the

development

of

virtual

HIV vaccines.

22

Finally,

23

carcinogenesis

24

the

25

biologicals

progress

and detecting

successful

experience that

are

in

understanding

adventitious with

actually

highly derives

agents,

and

purified from

tumor

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19 1

. cells. Discussions

2 3 -4

with in

the use of neoplastic the

Office

5

these

6

systematic

7

issues.

initial

which

was 'the

include

appropriate

11

the

12

models

used

13

criteria

to

14

the

15

meetings.

consider

data to

the

approaches

17

approach,

six

issues

18

and the concerns

19

slide. Icommittee

Of

23

points

generated

for

24

today's Issue

contamination

six

Issues

of

a

these

steps,

developing of

the

developing and discussing

in public

forums

of implementing These

are presented

were discussed

the

only

risk,

stages

in 1998 and again

21

originally,

of

of

developing

validity

were identified.

they

The issues

the

approach

In the initial

of the five

evaluation,

levels

or this

development

each issue,

your

consider

The outcome

issues,

to establish

issue

were begun

and evaluate

consisted

identifying

necessary

22

1996.

models to evaluate

16

25

discussions to

associated

substrates

in

This approach

10

20

cell

issues

of Vaccines

approach

8 9

regarding

in detail

and

this issues in this

before

the

in May of 2000.

issues

that

2, 3, and 5 will

we identified be the focal

discussion. 2

with‘the

includes possible

adventitious transfer

agent

of known or

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20 1

-unknown

viruses.

2

we will

include

3

encephalopathy

4

agents.

For purposes agents under

Issue

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7

activated

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information.

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And

5

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15

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17

was developed.

18

includes

19

riskposed

20

of a worst

21

available

22

and cumulatively,

23

the

with

aspects

of

what we're

The basic

assessing

case scenario

relative

24

risk The

risk

today,

the this

and

risk

of

Vaccines'

a defined

risk

evaluation

this

evaluation

of

where'

possible

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the probability

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of

Office

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cell

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adventitious

infectious

or recombinant

we will

of

possible

and/or

Issue

spongiform

residual

oncogenic

Now,

.defined

the

discussion,

transmissible

interactions

14

25

of

of today's

issues, risk

cumulative

using

individually data

to assess

of the product. concept

evaluation

and

implementation

will

be presented

of in

the more

NEAL R. GROSS (202) 2344433

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21 1

detail

2

residual

3

issues

by Drs.

Peden

substrate later

DNA and with

this

4

and Krause

morning

of

the

CBER approach,

6

Advisory

Committee

7

meeting,

the Committee

8

plan

9

discussion

10

substrates.

into

at

11

an

This

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the next

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neoplastic

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Maryland,

nine

our plan'was

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18

at the May Advisory

followed

the Office

21

Advisory

this

meeting cell

22

were

23

included

24

are transformed

25

no cell

divided

into

human cells

lines

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plan on

for cell

over on

in Rockville,

meeting, five

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and

last

Office

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of

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used for vaccine

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in a workshop

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substrates

Committee

of Vaccine's Committee

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20

this

of 1999.

of neoplastic

19

During

was implemented

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17

to the

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Vaccine

presented

workshop

recommendation

16

stage

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international

in September

15

discussion

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months

cell

public

document

agent

afternoon.

in November of 1998.

a draft

discuss

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and this

To implementthe

5

when they

hypothetical

the substance

of

at the May 2000 cell

substrates Category

manufacture Since

that

there

examples

1

are

include

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22 1

the

dipldid

WI-38

2

immortalized

and MRC-5 cell

by human telomerase Category

3

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cells

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16

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17

20

derived

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focal

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15

by

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be the

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293 cells

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gene.

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going

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cells

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and the HUT-

HIV virus.

were developedbased

difficulties

in

managing

the

regulatory issues associated with different types of _' -cells. Possible management approaches were presented for

each category. However,

22 23

review

the variety

24

raised

by cells

25

information

today of issues

in

each of

is available

time

doesn't

permit

and approaches these

that

categories.

in the transcripts

me to were This

of the May

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23 1

which

2000 meeting,

are present

Of these

-2 3

1 and 2 as examples

4

going-to

five

categories,

of designer

be discussed

6

meeting

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cellsubstrates

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you just

saw.

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9

designer

cell

substrates

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neoplastically

11

cellular

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13

design

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15

future.

16

present

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18

use.

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in

to

it's

and the

need

21

substrates

for vaccine

22

of factors

behind

23

cell

24

factors

25

the

replication

human

cells.

cellular

genes.

to express

of

Steve

the development

cells

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are a number

and use designer

development. of cells

bioengineered

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of

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Hughes

associated

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include

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talk,

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as

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19

into

of mammalian cells

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12

cell

Categories

the subject

issues

which fall

10

only

today.

And as I mentioned,

5

on the CBER web site.

These

to complement viral

vectors,

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24 1

.-

increasing

experience

I

and

2

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proteins,

4

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the

the

and use of bioengineered

7

serve

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Frank

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third

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missing

assist

the

morning,

Dr.

the

this

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23

by Frits

in 1998.

are transformed

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we'll

which by

line are

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in 1977. human embryonic

by a clone cells

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PER.CG

genes

vectors

retinal

on

vectors

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21

25

viral

talk

PER.C.6 cells, cells

and

vector.

Graham described

24

of vaccines

immunizing

viral

adenovirus

'The

16

active

have much more to say about viral

15

gene

out the development

defective

defective

In Aguilar

to point

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use

the

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in

biologically

development

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6

11

of

vectors

of HIV vaccines.

I should

5

viral

production

and hence

development

with

fragment

were described

beenverylittle'published a

considerable

amount

of

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25 1

'information

has accumulated

2

became available

3

will

focus

on 293 cells

in 1977, much of our discussion

The talk

5

morning

6

characteristics

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discuss

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17

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by a known mechanism.

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in animals

of

information

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in the

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available

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with

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are transformed

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in

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from mammalian tumors

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the

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the cells

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contamination

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the

with

similar

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contrast,

and

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this

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issues

with

Andin

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origins

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15

23

today

by Dr. Alex van der Eb later

of these

7

of

they

on 293 cells.

4

22

since

transformed

perspective,

this

additional

level

which might certain

are

of

be present

origin

to vaccine

type

are not

recipients.

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26 1

.-

The issue

2

with

3

neoplastic

4

tumorigenicity,

5

tumors

the

that

use

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cell

substrates,

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15

DNA or

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activity

slide.

associated of

17 18 19 20 21

to sustain

cells

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that

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in animals

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risk, cell

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with

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from

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with

to vaccine

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to grow into

between

development

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and

is

potential

assays

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rodents.

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of concerns

in particular,

into

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list

cell

is their

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for

the

proteins

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are

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unable to transform cells. This leaves cell DNA and . _ oncogenic viruses as the risk factors associated with cell

substrates

22

that

are tumorigenic.

In order

23

what

24

adenovirus

25

tumorigenicity

for

the Committee

we mean when we talk transformed

about

tumorigenicity

human cells,

of 293 cells

to appreciate

studies

are presented

of on the

in the next

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27

1

slide,

2

line

and they're that

compared

was established I have to

3 4

our

information

5

slides

6

the mouse obviously

are

9

Frank

a series

description

11

the cells

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think

14

million

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later,

17

animals

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ten million

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and

assays,

line,

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In the original

Graham reported

inoculated to

see

tumors

with

--

cells

this

but

--

and I

that's

ten

experiment

more detailed

inoculated

with

inoculations

100 million

per mouse,

ten

21

number of cells

22

somewhere

required

cells

per mouse,

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in the range The way that

24.

terms

25

dose at a 50 percent

to produce

cells

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TPD-50 value, endpoint.

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of

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That's

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of the

that

per mouse.

did a little

23

at

one done by

andtheyproduced

may be hard

of

making

what we're

tumorigenicity

We repeated

16

tumor.

a discrepancy

of 20 animals

cells

a cell

transfer

to the people

weaklytumorigenic, three

cells,

the

I became Lew is rather

of the 293 cell

15

20

for

Graham and two done by myself.

10

19

apologize

But in this

8

A-549

from a human lung

by computer

because

7

with

is in

producing

the number of

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1

cells

that's

2

of

3

comparable.

required

mice,

the

5

cells,

6

it

7

50 percent

which

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only

takes

about

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to A-549

from human tumors, to produce

tumors

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to say about

tumorigenicity

13

cells

this

morning.

The

potential

14 15

residual

cell

16

designer

cells

17

neoplastic

18

viral

19

latent -* -

viruses,

another contain

as well

22

viral

23

Latent

24

sequestered

and.DNA from oncogenic

oncogenes viral

can also genomes

in cell

induce in

in

oncogenes,

oncogenic

as retrovirus

with

DNA from

activated

genomes of

more

prepared

concern.

Clone cellularoncogenes in rodents,

in

transformed

associated

DNA in vaccines

can the

to have a lot

risk

represents

oncogenes,

21

tumors

of adenovirus

substrate

cells

20

inducing

Jim Cook is going

12

later

in

are about 1,000

are the 293 cells. Dr.

11

the A-549 cells

more efficient

than

viruses,

proviruses. caninducetumors viruses

tumors

and cloned in

rodents.

proviruses

retrovirus

DNA can be infectious.

Duetothese

25

are

of the mice.

10,000-fold

animals

in 50 percent

numbers

derived

1,000

Therefore, to

tumors

when you compare

is the cells

8

10

and

However,

4

9

to Produce

observations,

the possibility

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29 1

must

be considered

2

cell

3

activity

4

recipients.

substrates or

could

The talk

6

is going

7

the use of residual

to cover

designer

by Dr.

in detail

concern

contamination.

11

are

12

adventitious

agents.

13

the designer

cell

14

adventitious

15

neoplastically

16

Designer

17

contamination

18

agents.

to

is

possible

address

Due to their

substrates

agent

might

contamination

transformed

21

evaluating

22

agents.

23

might

unknown,

issues

designer

I'd

24

that

today

25

the

issues

morning

associated

with

like

we are facing associated

the

possibility

All

cell

of

substrates with

laboratory

origins,

represent

a risk

represent

possibly

of

they're

use of

will with

adventitious

my talk

a transition. the

afternoon

for

of

oncogenic

associated

conclude

with

a risk

latent

this

substrates

to

the use

because

specifically cell

with

and may be tumorigenic.

substrates

the

this

contamination

Dr. Krause in his talk

20

vaccine

associated

agent

adventitious

19

to

DNA.

10

with

designer neoplastic

genomes

the issues

substrates

cell

either

Peden later

cell

subjected

DNA from

transfer virus

The third

8

of

residual

infectious

5

9

that

by saying

By considering Adenovirus

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5

30 1

-transformed

2

with

3

substrates

4

over

cells,

the

first

of

that

the past

5 6

being

7

the

8

that

9

thinking

such as 293 cells, the

we've

years.

As these

cells

previous

they

way of

goes back

cell

this

12

confronted.

13

the possibility

14

come from

15

can

16

technology

17

vaccines.

be

However,

18

ability

19

objectively

20

types

of

21

about

their

22

vaccines.

23 24

assist

25

invited

substrates ways of

the

determine

think

be

presents

Those rewards

will

the

that

benefits

application

of

of

safe

facing

us

molecular

and effective

today

available these

to produce

safe

is

in who

this

to

on these

data

tell

us

and effective

the end of the slides.

Committee

individuals

must

also

what

that's

of situations, that

the data. that's

CBER and the those

types

rewards.

challenge

potential

I

of from

future

transition

development

review cells,

to

to maximize

by

the

The

category

cell

risks

this

of future

to

Committee

a transition

decades

presents

obtained

the

about

most of these

transition

the

the

cell

substrates.

As with

11

'with

represent

four

confronted

neoplastic

fall.into

thinking

over

about

novel

discussed

three

tumorigenic,

10

truly

we're

review,

have

To We've

introduced

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31 1

themselves

to you whose work qualifies

2

with

3

complementing

cell

4

to review

relevant

5

answer

6

opinions

7

addressed.

sufficient

experience

the

data

the

Before just

take

this

10

that

they

have used to assist

11

the

12 13

try

to answer

concludes

they

speak,

I'd

them for

the Office

my talk.

much, Dr.

16

us to continue

17

Lewis.

That provides hearing

our annual

like

to

the time

discussions. I'd

be happy

also

about

reminds

visual

a useful this

to

very

setting

for

some of us that

screening

it's

20

ACTING CHAIRMAN DAUM: opportunity

22

scheduling

23

Alternatively,

we can get some more information

24

table

initiate

and then

there

Is there

a little are

bit

We do have

21

if

time

test.

(Laughter.)

run behind

you

issue.

19

25

be

any questions.

It for

to

of Vaccines,

ACTING CHAIRMAN DAD-M: .Thank

15

to

their

need

in these

raise

offer

.that

to

viral

Committee,

to

to thank

and the public

This

14

18

opportunity

the

and

begin

9

Committee,

before

issues

they

defective

and the issues

questions,

regarding

8

with

systems

Committee

them as experts

here

Committee

the

in terms

of

questions. on the

discussion. Committee

input?

Dr.

Goldberg,

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32 1

and then

Dr.

DR. GOLDBERG: Yeah,

2 3

tumorigenicity

4

tumorigenicity

5

you give

6

distinguish

where

me some feel these

for

sorry

9

mice

--

ten

another

to

rates

that

you have -- I can't

suggest

that

13

distinguish.

14

the

sixth

any tumors in

you

15

other

16

make the distinctions

really

four

information

with

give

you're

in

bit

19

You're

20

5'0?

of

a hard trying

21

time

four

you do that,

hearing

23

how do you feel

24

four

25

really

mice

but my concern that

estimate

animals

can't

for

to bear

I'm

what

what

on this

having

to

you

a little

were

how we calculate

I think

the TPD-

really

experiments

dose levels

the TPD-50 with

saying.

I do know how

or my question

based on these

at each of these

I do would

what the TPD-50 is?

DR. GOLDBERG: No,

22

nude and in

me some feel

I guess

to understand

I'm

of four.

four

bringing

about

DR. LEWIS:

18

can

you can

one experiment

you observe

So can you

17

of

see.

And you know, any calculations

12

of

in the nude mice,

how you feel

observe

experiment

11

the

table

levels?

you don't

at

on your

show

YOU

For example,

8

just

in the 293 cells

7

10

Griffin.

that

any certainty

is: with

you can to make

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33

,...,

1

.a distinction

between

--

2

DR. LEWIS:

3

DR. GOLDBERG: --

4

to the

sixth,

for

5

Okay.

used to do this

7

we did

8

These assays

9

they

Basically,

10

expensive,

11

deviation

12

of a log.

done

in

four

and each time of those

ten

times,

mice,

but

they

assays

with

based

14

obtained

15

this

represents

16

type

of information.

mouse

19

numbers

cells

that,

on

nude

plus

the

an accurate

were

repeated

are basically

the

ACTING

21

DR. GRIFFIN:

puzzled

23

this,

by the

are

standard

or minus

.6

cells

is

we that

reflecting

this

and many of the

least

twice,

and

the

same.

Well,

same table.

The difference A-549

at

that

confident

way of

CHAIRMAN DAUM:

and I got a little

24

the

mice

information

we are reasonably

20

22

cells.

and each time

The data on the 549 cells

17

293

that

were done the

was about

was

Okay? So

18

that

of titrations

transformed

12

were repeated

13

25

the data

came from a series

on Adenovirus

were

and ten

example?

DR. LEWIS:

6

ten to the three

Dr.

I guess

Griffin. I was being

And maybe I just clued

in what you just

between

one is Ad.

missed said.

the 293 cells

5 transformed

and

and the

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34 1

.-.other

is Ad. 12 transformed?

2

DR. LEWIS:

3

DR. GRIFFIN:

4

DR. LEWIS:

5

line

6

oat

7

established

8

tissue

9

are a cell

10

was established cell

from

that

in nature,

is that

The second

point

15

Adenovirus

5 transformed

16

This

17

cells,

18

as adenovirus

true

20 21

is

both

Adenovirus

in

-would

define

characteristic

it

cells

into

to

are

number of

produce

tumors.

transformed

hamster

--

one point.

a large

tumorigenic,

23

ACTING CHAIR&

24

I'd you very

they

mouse

cells,

as well

a categorythat,most

DR. GRIFFIN:

thank

They

human cells.

22

25

the

to be made from

that's

takes

of Adenovirus

like

in

tumor in the human.

adenovirus

as weakly

an were

transformed.

yes,

5 transformed

They fall

And they

in how likely

transformed

a cell it's

human tumor

So the point

that

are

--

from a human tumor that

Well,

14

19

Okay?

the

differ

DR. LEWIS:

is

cells

I believe

a spontaneous

cells

13

A-549

developed

DR. GRIFFIN: this

have different

They are not virus

line

11 12

No.

carcinoma.

directly

developed

And they

from a human.

(phonetic)

culture.

No.

people

and this

5 transformed

is

a

cells.

Thank you. DAUM:

to move on then

much, Dr. Lewis

Okay.

Thank you.

at this

point

--

-- to Dr. Steve Hughes'

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35 1

presentation,

2

Vaccine

entitled

Development:

,3

Dr.

Concepts

may challenge

people's

7

Since

9

quickly.

this

by Andy,

10

I'll

11

course,

is

12

differ

from

13

transformed

14

spontaneous

transformation

15

cell

and that

16

an animal

17

culture,

18

rodent

19

cells

undergo

20

don't

understand

21

ability

22

of their

and cells

cell

and

it's

an embryo

of

24

immortalized

25

was just

mentioned,

fact,

the

past,

that

of

alters

in culture

way cells

from

cells

from

them in

characteristic

and biological

or immortal

of

with

and passage

change,'which

clearly,

other

in

lines in

some period

some sort

The

this

consider,

means you take

to grow permanently

23

ably

has been used to establish

after

physical

to

cell

a particular

that

so

go through

basically

simply or

been

substrates,

permanent

cells,

usually

and

question

how designer

I

once again.

has

try

the

other

state

subject

Basically

lines,

for

is somewhat smudged.

optical

Thank you.'

introduced

Substrates

Hughes.

6

8

Cell

and Issues.1f

DR. HUGHES: This

4 5

"Designer

of

passage

the

we still both

and alters

their some

properties. that

cells

have

have been derived

tumors

taken

from

been is as either

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36

1

..humanS or

2

established

3

these

4

animals,

5

establish

6

researchers,

7

laboratory.

animals, directly

tumor

in

cells

And it's particular

very

disadvantage,

11

taken

place

12

c,ells

that

13

that

don't

14

transformed

in

notion

cells,

have

the

makes everyone

17

cells

18

specific

19

that

is

we don't

forever

it

of

23

idea,

24

designer

25

these

what's

meant,

cell two

of

these

nervous

about

these

they

have

that but,

in fact,

is that

differ

is

think

types

to

normal

differentiates

fact them. cells

with.

that told

of

something

comfortable

that

I

the very

the

as Andy has just that

cells being

has changed

from

reasonably

cells

either

that

them,

substrate

types

about

have

in culture.

And as an alternative

22

is

things

know what it

feels

has a

in neither

the

necessarily

know how they

everyone

that

it

the

of what changes

properties

a little

wrong with

.We don't that

not

in

but

is

what

And so one of

in

basic

them from the normal

or growing

16

that

routinely

convenient,

differentiates

15

21

these

lines

use

and that

case do we have any clear

passaged

have been used to

of cells

myself,

as

can be

some cases

serially

methods

a number of types such

and in

then

of these

10

20

some cases, these

culture,

are

and both

8 9

and in

kind you, it

of by a from

one now can take

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.

37 1

specific

--

2

specific

DNA segments

3

derived

4

properties

which

from

that

of normal

either

the cells

and some particular of what's

that

one might

have.

There

One of

issues

there

is

is

whether

this

actually

has some sort

that

specific

material

using that

So there

the

degree

in

is

there

to

which

would a vaccine

you're

this

is

it. capable

culture,

it

as you've

is some reason region

to that

of that. worry

about

preparation

going

is actually

of added

with

and in fact,

potential

SO you really

25

it's

in the case of the adeno early some oncogenic

24

when

to grow forever

all

question

DNA segment

18

vaccine

the

this

if

cells

you're

some

are issues.

associated

may have oncogenic potential, / just heard discussed by Andy,

the DNA if

handle

eliminate

of risk

of

is

course,

DNA that

16

there

is

on.

that

that

it

we have at least

the worries

think

we now

us some particular

of

the

growth

differently.

does not,

causing

the

what agent

This

the

virus,

change

to behave

feeling

--

from

and in so doing,

gives

going

correctly

derived can

cells,

Of course

23

spelled

in which we understand

is causing

idea

not

that

And that 8

have

cells,

have something 6

I

carrying with

the

a question

of

concern,

and

to use. still

a serious

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38 1

you'll

hear more about

2

be pleased

3

discussed

4

should

5

and

6

assessment

7

defined

to

later

today.

you one of

the last

time

this

the

the

to

FDA

of

-try

and

what

amounts

the

I'm pleased has reached

the point

10

will

11

exactly

12

DNA segments.

what

the

14

issue

is

15

cell,

16

culture,

17

be

18

associated

whether

it's

whether

infected

is

at

with

20

tried

to allude

21

is both

22

and,

23

determine

24

agents,

virus,

both

of

diploid

there

and establish some defined

there

is the that

any

permanent

in

fibroblast

have

these

other

cases,

in the DNA, in part what sorts

trying

can agents

what.agents, what adventitious

as. I

the question

of things how it

in particular

for

agents tried

might

just here

pose risks

to understand

And so what I've

25

is

can

using

and that

is to say,

that

a norma.

understanding

secondly,

cell

of

interaction

from

that

the NC1

it.

And in

19

a

that

mentioned,

agents,

there

DNA segments.

to try

least

was

quantitative

terms

funded

studies

it's

with

in

oncogenic

where it's

risk

of adventitious

.a more

risk

As Andy has also

13

between

to say that

be some quantitative

that

group met was that

get

of defined

I would also

things

be in a sense a collaboration

8 9

tell

that

is we can

adventitious be present.

to say is that

the

NEAL R. GROSS (202) 234-4433

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_-.

_

*=

.*_/--*

._ll_

..vx~17-.-

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---------.

---

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39 1

_ issue,

I think,

2

of

risk

3

are reasonable

from the DNA is at least

assessment,

and I think

ways of defining One can take,

4

that

particularly

what

6

those

DNA segments;

one can take

7

those

DNA segments;

one can inject

8

models,

9

on that, idea

11

risk.

dealing

and one can define

of what it

13

one of the nice

14

and biotechnology

15

of looking

16

and you'll

17

considerably

18

sorts

19

of doing

for

the

about

is that at least

22

problems,

23

should

these

measure, in terms

adventitious

acid

some of the

agents, biology

bearing

a little

are under

tools what

and

sorts

we go about

of

trying

And I think

24

it's

and based

modern molecular

than

So the question .have

animal

later

I intend

ways

agents, today

to discuss

consideration

in the

as ways

this.

20 21

them into

facing

nucleic

I think,

that

amounts .of

we now have much better

more detail

of things

one can take

defined

case of

are.

one knows

the oncogenicity,

we're

things

hear,

with;

one

actually

if

some reasonable

is that

And in

12

25

one is

one can get

there

what the risks

5

10

DNA segments

in part

going

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that

things

we

should

that

have we do.

we

these How

to be as safe

as possible?

one of the things,

and 1,think

to come up in considerably

more detail,

is

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40

1

_ culture

2

cells they

history.

You would

have come from. have

about

spent

where your

are

briefly, if

worth that

time.

It's

teenage

children

one just

and it's

are

about

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10

fact,

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11

serum turns

and,

for

viruses

14

there's

another

issue

that

15

discussed

16

think

17

substrates,

18

sense. directly

from

19

idea

one of

20

deriving

21

to passage

does matter but

not for

it's

cells

cell

23

which

24

favorite

25

element

is

one which stories, of

I don't

risk

be in the

agents

like

believe but

has been actually

designer

are derived

material,

and that's

traditional lines

BSE

And I think

that

the

in

I cell in a the

methods

for

from human tumors,

is

in mice.

And there's

22

both

even so much for

particularly the

that,

consideration.

detail,

tumor

only

history,

earlier

as well.

substrates

I

obvious

or culture

for

any particular

that

probably

I believe,

course,

that

things

of immediately

passage

is true

13

in

of

to be a substantial

And this of

worrying

of the serum and what might

out

12

like

go at night.

although

to,

the

to know where

of

a cou.ple

not be sort

thinks

know where

like

sort

discussing,

might

to

You would

A.nd there think

like

a particular

makes 'for

consideration

one of

that

suggests

here

that

that

people

John there don't

Coffin's is

an

always

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1

. consider.

2

Mice,

3

of

4

endogenous

5

cells

of course,

endogenous

contain

retroviruses,

retroviruses

derived

and

in non-rodent

9

example,

mice

is

human cells

that

10

actually

11

viruses

12

to actually

that

for

provides

13

kind

of culture

15

that

one would

16

you'd

17

or a primate.

quite

are very

20

give

21

culture

22

necessarily

23

agents,

but

24

history

what

25

look

when,

passaged

through

nude

--

opportunity these

are

in non-rodent

really

possible

the

cells

important

substantial

not normally for

in a cell

--

so

out all

sorts

derived

from a human

I think

of

to much

having because

possibility

we can understand

if

agent

would be one

and certainly

not

rule

in this

think

of considerations,

consideration

history

actually

to add an adventitious

SO these sorts

19

happens

viruses

history

have to look

18

that


And it's

14

replicate

a wonderful

1ik.e to replicate infect

these replicate in

cells.

are

xenotropic

that

of

and some actually

And one of the things for

families

some

preferentially

from mice,

preferentially

several

I think, we have to a defined it

will

of adventitious we know the culture

adventitious

agents

we should

9

for. NEAL R. GROSS

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42 1

And one of the problems

2

adventitious

3

acid

4

you only

5

what to look

agents

technology

that

find

And

7

perhaps

8

part

9

particularly

for

will

the things for,

6

with,

it

10

issue

11

phenotype

the

you look

final

problem

of

the

idea

part.of

yet

stability

you know

the

I

is

the problem,

the

sure

I

have

a

resolve,

is

the

genotype

or

the

of the cells.

12

And the

13

actually

14

talk

15

lines

by simply

16

there

is

reason

goes back to the

with

that,

in

18

spontaneous

19

properties

20

passage

21

change

in

I introduced

course,

not but

cells

can change.

in

cell

in

fact,

transformation. only upon

culture

They don't

the

cells,

that,

as spontaneous

of

the

that

a consideration

culture;

transformation, of

is

you can derive

passage

AAnd

this idea

fact,

such a thing

17

is passage

upon

there the

prolonged

have to change,

but

can occur.

22

Now,

23

phenotype,

24

genotype

25

is

think

not

how to of

and if

that

I'm

nucleic

much easier.

thing

that

the

for

many viruses

for,

makes your,job

the

good

example,

be used for

the most challenging

of

in searching

for

that sure,

has altered Cell

means

that,

in

and. probably

the

during lines

do

the passage change

fact,

the

underlying

of the cells.

upon

prolonged

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43 1

.passage

2

arises

3

the

4

culture,

5

with

6

confident

7

has the desirable

8

we put

9

given

10

in if

cell

culture,

that

is true,

properties

that

in

which

of

the

fact,

that

that the

how do we gain after

is

the cell

lines

seems to

substantial

13

course,

one of

14

people

is simply

that

15

a relatively

16

course,

17

spontaneous,

18

low passage

cells,

19

taken

seems to be better.

20

there's

old

these

place

the

a change

22

to at least

23

might

at

24

using

some sort

25

expression

quite

line

that

the changes a long

time,

me to

be one of

of

that of

tissue

and of culture

have been passaged

times,

of genetic chances

the

and

that,

of

appear

to

be

accident, that

by using

some change

The hossibility

has that

seems to be less.

begin least

we're

how do we know

changes

But the final

21

cells

cell

for

standards

number

some sort

the

we need to consider,

to use cells

because

it

in

changed?

12

small

of

can change,

hasn't

the

that

and has only

we passage

requests

confidence

to say if

and then

And that

then

some period

made a designer

properties

line

question

the properties

That

we've

cell

11

cells

match

we began.

in

and so the

to think

in of

thing

that

about

some cases

give

regulatable

of the gene that

is

I think the

idea

that

consideration

system

causes

we ought

to

the cell

drive

we to the

to change

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44 1

.its

properties. And if

2 3

have

some sort

4

that

we can turn

5

off

the gene that

6

the cells

7

--

8

in fact,

9

cell,

of

promoter

on and off we're

--

that

the agent if

properties

ought

which

that

And I mean, out

14

want to use necessarily

15

idea

16

a way to regulate

17

interested

18

negative

19

nucleic

that

as an idea,

I think

in,

21

about

22

passage

23

properties

24

agent

25

we've

the

or the

-- ~'rn sorry added,

or

I'm

is

but the

somehow to find

of the

gene you're

some sort

of

protein

dominant

level,

at

the

promoter. I think,

can we determine that

we think

is

is,.

in

the

fact,

think

after

some

changing

the

-- the gene,

the designer

have

been

there

throwing

You may not

we want to,

NEAL,R. (202) 2344433

of the starting

inducible

that

cell

the

promoter,

here

at the

the agent of

then

may be that

it's

is can we.validate, that

cells'

expression

either

The idea

20

off,

as a solution.

whether

level

of the

an inducible

the

added is,

or transformed.

is central

effect acid

it

not

is the cell

the properties

back to that

was not permanent

this

that

on and causing

the gene we've

gene

to fall

if

we

on it

we can turn in that.is

then

changes

that

has a switch

so that

if

that

13

example,

interested

is,

we switch

for

that

to be transformed,

I'm,sorry

cell,

we imagine,

responsible gene that

some additional

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45 1

changes

2

influencing

in

the

the behavior

4

that

idea will

5

I'll

stop,

6

answer

and

giving

be important,

and if

phenotype

that

are

some consideration

to

of the

And I think

3

there

cell.

and I think

at that

are ques.tions

I'd

ACTING CHAIRMAN DAUM: Okay.

8

questions

9

Hughes' for

point

be happy to

them.

7

10

genotype

at

this

time

to be focused

presentation.

more general

There

discussion

DR. COFFIN:

11

raise

actually

didn't

13

one is

14

vaccines,

that

15

actually

16

by some sort

17

that,

18

particularly

19

picked

up an endogenous

20

early

genes of adenovirus.

and that contribute

aware,

cells

is

potential

the

that's the

22

you're

23

there

24

by others

25

are somewhat different

will

for

retroviruses

avoided

of

time

an issue

you

of

of the

cells

and because

viral

virus

of

could

a cell virus

I deliberately,

that

when

growth

that

xenotropic

for

on Dr.

to

itself

and the consequences an issue

be consideration'of later

there's

these

DR. HUGHES:

21

be plenty

the

comes up particularly

of recombination

if

mainly

genes to the vaccine

and I think

like

later. Steve,

12

considering

will

I'd

issue

of

arise, line

has

or with

the

as i'm

sure

both because

I think

the recombination

issue

I believe adenovirus,

that

the issues

which

I think

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46

1 2

supposed

-we're

to

And I think

4

the

5

pertain

6

the discussion

7

omission.

mechanics

of

recombination are a bit

today,

and

of recombination

and

10

one,

and

11

consideration

12

can do to cells

13

sense what the cells

14

the viruses.

15

that

the

that

think

issue

we

but,

that

that

and Dr.

18

DR. KIM:

20

,are shown not

21

oncogenic?

22

a real careful

what the viruses

in the cells

can do to

Thank

you,

Dr.

Hughes.

Dr.

substrates

Coffin

in some more complicated

or things

17

19

is

very

ACTING CHAIRMAN DAUM: Coffin

as Dr.

give

of not only

in fact,

was a deliberate

he raises

should

to issues

as they

beyond the scope of

So that

But I certainly out

Kim. Are

on the horizon

there

any

designer

or on the radar

to be oncogenic

DR. HUGHES:

or

less

cell

screen likely

I'm not qualified

that to be

to answer

that.

24 25

on

particularly

we have here.

points

23

the issue

to retroviruses

8

16

focused

retroviruses.

3

9

be

ACTING CHAIRMAN DAUM: Would you like try,

in looking

for

answers

to this

to

question?

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47 1

.-

(Ltiughter;) ACTING CHAIRMAN DAUM:

2 3

recognize

Sorry,

Dr.

5

DR. AGUILAR-CORDOVA: Yes.

6

the

7

tumorigenic

8

events.

9

just

10

still

Aguilar-Cordova.

transformation

of cells

there's So if

one has only

be there

on that

DR.

11

and for

some old data

make ten minus one

is

14

multiple

15

things

16

chemical

17

have been confirmed

18

that

19

the required

20

it

is,

21

if

we make any of the changes,

that

agents

anything

if

transformed

24

example,

25

only

not

we now believe changes

or

viral

change,

event

may

sure

it

certainly

are needed,

but

many of the

whether

they're

and these

studies

manipulation

of mice,

moves us one step

be it

I

most tumors

two,

three,

that

you do bring

cell

you

of changes, that,

closer

can

show,

mice,

get spontaneous

to

whatever

by doing

the

and that

closer

five,

to the list

in mice by the p53 knockout

a single

that

for

by genetic

phenotype,

of

that

agents,

we do that

change,

wouldn't

but

as tumorigenic,

number,

a series

quite

question,

we add any one thing

the

23

your

we regard

to become

showing

I'm

13

genetic

about

genotype?

precisely

case that

a cell

and the oncogenic

HUGHES:

understand

You talk

one agent,

12

making

I do

you are number two in line.

4

the

but

which

tumors

by to a for have

at a very

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48 1 2

.. high

rate

because

one

And I think

4

we would be nervous

5

cells

6

actually

from

7

substrate

was, actually

8

things

9

human towards

safety

has

that's'

about,

the sort

been

of thing

and of course,

the

DNA, depending

necessary

to

deliver drive

the transformed So the single

11

the

cells

12

makes us feel

13

I

14

safe.

more than

don't

think

it

in

an animal

Then Dr.

there

one.

So I think

are more thanone,

means that

things

were

DR. MINOR: The tumorigenicity

18

in

rodents

19

clearly,

but

20

species

effects;

that

21

out

things,

that

22

tumorigenicity

for

very,

is

it

very

good

possible

you think

25

it but

perfectly

please.

if

you took you

ranking

assays done

technical

that

there

reasons

are

actually

the immune response

would

find

in a different

I mean, how relevant

23

a

Kohl.

17

of

or

and some of

ACTING CHAIRMAN DAUM: Dr. Minor,

15

cell

one of the

one is not good,

that

the

phenotype.

may have had more than better

you could

on what

a cell

that

some of the

may have more than one change so that

10

24

of

removed.

3

16

layer

a

different

species?

are

the

rodents

do

to a human situation? DR. HUGHES:

I

think,the

answer

is

-- and

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49 1

.this

by

is

2

experiments,

3

So

a

in general,

can only

have to sort

you

5

from.chemical

6

in fact,

7

cases.

the

choice

10

doing

the

11

the

12

happens

13

all.

is

strong

And I think

that's

in

some sense

that in

in

15

exact

sort

16

doing

the

17

humans,

1%

try

humans,

and not

19

data

I think,

species

effects

a concern,

but

I think

is

between

in which

perfectly doing

experiments

in

you have

reflect

the

terms

of

in rodents would rather

about

the

that, in some

what

.experiment

I have some reservations

you mention,

and worry

good data,

rodents,

may not

I certainly

the

be done in rodents.

experimentally

it

And while

14

because

to make one believe

are very

experiments

worry

enough

carcinogenesis

there

8

speculation

of extrapolate.

But there

4

9

definition

worrying

and applying have rodent

extrapolation

than

at

of the about it data

to and

have

at all.

20

ACTING CHAIRMAN DAUM:

21

Dr.

22

DR. KOHL:

23

ACTING CHAIRMAN DAUM: Dr. Myers,

24

DR. MYERS: I guess I have two questions.

25

no

On the

Kohl and then

confidence

of

Thank you.

Dr. Myers.

That was my question.

the

stability

of

the

please.

genome,

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,.

1

would

2

were excised

you be more confident

that,

5

relevance

6

mice?

and

And the

second

that

could

is

of the

7

excise

the

to

you can interfere

with

11

DR. MYERS:

12

DR.

14

trying

15

is

16

necessarily

17

technology,

18

a technology

19

ask the

not to get

not

even but

but

20

If

that, you then

21

'the expression

22

cells

23

does that,

24

as you would.expect?

25

And if

towards

the

in fact,

point

I'm

I so

that

interfere

change'the

really at

technology to

will

in a sense,

permanent

is

you want to look

allow

you're with

you think

you can do that,

either you to

posing.

or obliterate is driving

tumorigenic behavior

or

a precise

to somehow develop

of the thing this

but

technology

yourself

or technologies

question

probably

segment,

precise

limit

to be able

it's

Knockout

the to

the

out?

at is what I think

necessarily

about

up the experiment

Knock it

easiest,

to

the expression.

HUGHES:

the

related

to nude-nude

think

are ways of setting

probably

us

I don't

think

13

tell

DR. HUGHES:

9

there

is

tumorigenicity.limited

feasible

segment

was lost?

you

technically

that

designer

question

8

10

the

and the tumorigenicity

3 4

if

the

phenotype, 'of the cells

I think

you're

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_ .il

-.

.

-

-quite

confident

that

there's

2

nothing

substantial

3

I think

actually

4

mouse. question

5

deferring

6

much more of an expert

7

than

in terms I'd

feel

I understand

it,

11

relevance

to normal

12

the

13

the

14

potential,

15

immunocompetent

system

,16

immune response

which

argument

the

to the

nude

comfortable

Don Blair,

probably

who I think

is

in nude mice

mechanism

cell

be that in

and the

be lost

the

failure

22

presumably

23

in

because

potential,

tumorigenic

nude, to

could

25

you know,

by demonstrating. you've

shown

be tumorigenic

at

arises

the

in

an

from

the

at

some

some stage,

it

you know, the demonstration a immunocompromised does demonstrate

as opposed

is no potential

to

Dr.

Blair.

Dr.

Lewis,

of

system

is

there

is

that

no potential

or what

at all.

ACTING CHAIRMAN DAUM: clarifying,

animal have any

and I guess, at least

is,

or be modified.

tumorigenicity

that

is

presumably

So I think,

important

I guess the question

situations,

tumorigenicity

21

24

that

would

18

.20

I --

is in an immunocompromised

fact

the

more

on or

know that

of responding

Well,

does the

19

I don't

going

I am.

10

17

else

on tumorigenicity

DR. BLAIR: if

on.

to my colleague

8 9

going

nothing

then

Dr.

Thank

you

for

Aguilar-Cordova.

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52 1

DR:LEWIS: question

about

tumorigenic,

only

occurs

three

7

transform

8

tumorigenic.

9

a telomerase

11

understand

12

literature,

gene, right

that

But those

those

it

in the

takes

and SV40 to is,

with

cells,

in

fact,

hTERT with

as

far

as .I

aware of in the

tumorigenic.

so far

as a designer

15

cells

now from what we're

are not

13

16

to a cell

and

with

that a,

not

aware of

cells

telomerase,

cell

are

we're

suggests

You can immortalize

10

14

data

genes,

a normal

that

to Dr. Kim's that

immortalizing

gene

different

cells

system

by

human telomerase 6

in response

immortalized

the

which.that

Just

nobody

cell

has

substrate

proposed for

ACTING CHAIRMAN DAUM:

one of

our attention. Thank

you,

Dr.

Lewis.

17

DR. AGUILAR-CORDOVA: Myquestionactually

18

follows

19

what

very

well

I started

20

on that,

a follow-up

on

to say. So if

21

particular

22

SV40 T antigen

23

-background

24

complementing

25

and it's

it's

event,

of

a series

whether

it

of

be the

or myc, be oncogenic the

cell

that

events,

it

would

telomerase, depending

hits

so that

a the

on the they're

oncogenes? And

I

guess

that

begs

the

question

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to

53 1

whether

any cell,

2

depending

3

normal

4

example.

It

5

immortalized

perhaps.

tumorigenic

on what the target if

it

only

many, many hours,

8

cells

9

example,

that

could

11

by change

in

12

change -in behavior

believe

15

produce

16

vivo,

17

a time

18

suppressor

or

there

if

the adding in

it

transformed

growth

a single

gene.

are certainly

takes

removing

23

in some sense keep track,

24

to

25

fact,

additive

by,

for

levels

of

either pattern,

to

changes

to

phenotype

in

oncogenes

one at

or ablating

tumor

does substantially even

change

if

the cell

it's to its

not full

phenotype.

So I actually

earlier,

at

reasons

multiple

tumorigenic

actually

22

say

high

in

the properties of the cells, .sufficient necessarily to drive Frank

delivered

of individual

some cases

genes

microscopes

very

change

of adding

even

for nor

in many cases see the effects

the Frank phenotypic that

through

give

morphology,

that

end myc.,

Those of us who have spent

that

So I think

14

It would appear

tumorigenic,

oncogenes

retroviruses

expression

21

appear

single

10

is.

is any safer

an activated

days peering

had

13

cell

wouldn't

7

20

has

DR. HUGHES:

6

19

or normal,

that as far

believe

it

and I also because

is think,

these

as we know

important

as I tried

things in

to

are,

humans,

in that

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1 2

-providing

one or

oncogenicity

Wo

steps

is something

3

One

could

in

the

we'd like argue

chemicalcarcinogenesis

5

the changes

and the long duration

6

development

of the tumor after

7

the chemical

8

the chemical

may have changed only

9

and the rest

must occur

10

But

11

substantially,

12

need to worry

that

many

that

you see to the

much, Dr.

the fact

Cordova

--

18

will

19

development

tell

that

one or two things,

spontaneously

later.

enhances are

the

things

risk that

we

Thank

you

very

Hughes.

a very

17

to

about.

I think I was

exposure

represents

those

that

one or two of

the initial

still

the

I think.

cases

exactly

ACTING CHAIRMAN DAUM:

15 16

actually

and I think

13 14

provides

of

to avoid,

in

4

insult

direction

we'll

helpful

move on, if

presentation,

to

I hope I'm not butchering us about

adenovirus

you would. Dr.

That

Aguilar-

your name -- who

biology'as

related

to

and use of adenovirus

vectors.

20

DR. AGUILAR-CORDOVA:

Can you hear me?

21

Yeah.

22

general,

23

that

24

use as vectors.

generic

So

background

we can use this

25

I'm

for

just on the

further

So adenoviruses

going

were

to

give

adenoviruses

discussion

identified

a so

and their

in

the

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55 1

early

2

tissue,

3

associated

with

4

lay

referred

5

inflammations,

from

'50s

thus

group

6

an adenoid the

name

an adenoid

adenoviruses,

some fairly

It's DNA encapsidated

8

and there

and

common illnesses

as the

it's in

"common cold,"

9

nature.

composed of a linear, in a protein

the

some eye

double

shell;

are many different

10

from

et cetera.

7

types

stranded

hasno

envelope,

of adenoviruses

in

They are primarilyclassifiedbasedonthe

11

organism

12

Mastadenoviruses

13

come from mammals and those

of origin,

and so there

Hopefully

15

(Laughter.)

16

DR.

17

characterization

18

terminal

19

and,

20

Serotype

21

there

22

hemagglutination,

23

blood

that

nobody

in

knob in the fiber the

has slides.

2, Serotype

are many other

The

and

different

of

.the

and hexon epitopes you'll

hear

5, the most commonly

binding

further

antigenicity

protein

serotype,

that

come from birds.

else

the

groups:

those

AGUILAR-CORDOVA: is

thus,

are two major

and the Aviadenoviruses,

14

serotypes,

of the fiber

about

used,

and

and also

by

protein

to red

cells. And

24 .25

tumor,

adenoviruses

it

turns

out

that

have more tumorigenic

some groups ability

of

in rodent

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56

1 2

None

have

later

on,

. cells. probably

4

hexons,

5

pentons,

6

fibers.

7

what constitute

I2

for

and

as you

a double

10

repeats

11

and here's

the

see,

serotype that

list

important

14

condense

.it

that

the

17

here

18

critical

for

19

and thus

the majority

20

adenoviruses

are vectors

21

been deleted

and replaced

is

ElA

these

little

and the hexons are

by two terminal that

terminal

genome,

protein

is

of the genome and to

from both and

ElB.

is linear

strands. The

expression

of other

ITRs,

inverted

include

five

mentioned,

region,

that

in whi,chthese

heard

these

are

are used in

two genes have

by the gene of interest.

terminal

the

but

genes' of the virus,

of the vectors

early

as well,

What you've

El

There are two origins

22

I just

and 12

it.

does express

25

has

go with

stability

it

units

phases

240

of them.

16

24

has

of the virus.

The gene structure

the

It

genome flanked

to keep the

15

23

hear

icosahedralproteinbasethere

Primarily

13

will

in humans.

20 triangular

and some proteins

12

you

icosahedral.

can

stranded

the

as

and the pentons

Inside is

is

each of

The fibers

8

shown,

to be tumorigenic

The virus

3

9

been

of replication.

repeats,

genes,

E2 region,

In

transcription

the ElA and ElB that the

E4 region,

and

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.

57 1

the

E3 region.

2

these.

1'11

a little

There are two delayed

transcript

that

includes

The ElA, 'is

talk

host

activation,

'6

activate

the

7

S phase,

and that

8

the

other

cell

growth,

11

in part

12

regulate

13

retinal

really

virus

hosts

16

E3, there

17

is protection

18

regulates

19

to be immunogenic.

two proteins,

23

and

proteins,

25

regulation

of most of

expansion,

induces

proteins.

proteins.

from viral the ability

It's in

It's

p53

and

involved

in

recognize the

E4,

that.has

of MHC Class

has miscellaneous

of transcription,

are

this

it

down

the virus

is that

it

down

1, and thus

proteins

there

that

and thus

of the cell

the other

particular.

believed

infection,

the expression

and it

genes that like

DNA replication,

are four

And

24

to enter

and it

One of the down functions

can't

is

(phonetic).

reproduction,

host

does

to some of the cellular

cycle

15

22

and this

a transcription

E2 has three

regulates

five.

genes;

blastomagy

21

late

and we know no what ElA and ElB do this

cell

20

'this

and induce

activates

by binding

14

and one major

are two proteins,

transcription

ElB is also

10

more about

one through

and what

adenoviral

9

early

late

there

bit

going at

activities,

MRNA transport,

least

the

on. four such as and DNA

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58 1

.-

replication

of the virus.

2

The late

3

They're

4

itself

mostly

involved

and the

6

really

7

in

stabilization

two faces.

8

includes

9

cell,

the

first

of that

life

cycle

hours

absorption,

of

12

definition

start,

13

so, and that

14

virions.

15

that

Traditionally

17

per

18

produce

19

more than

20

than

cell.

late

now that

them in the

laboratory,

So wild

ten

there

and

events

by

18 hours

or

is the construction

about

the

genes.

in the next

We.know

that.

into

of the early

it was said there's

that

core,

And we have approximately

16

and

virion

the

is

which occur

of the virus

the

and that's

them.

core.

infection,

begins,

is when there

of

of the virus

events,

after

and translation One

five

and replication

penetration

disassembly

11

are

There are early

six

transcription

there

in the structure

The viral

5

10

genes,

of new

to 50,000.

10,000 virions are

--

when we

we can produce

type probably

also

a lot

does more

that. Now, the next

21 22

the

virus.

23

gene transmission

24

gene therapy?

25

How does

step,

of course,

one use the

and, therefore,

And

there

are

virus its

two

is to

optimize

potential

key

that's

factors

use in

of

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1

adenoviruses

that'actudliy

2

variance

3

can package

up to

4

when we take

out the ELA or the ElB genes,

5

a little

of

6

five

as good,

bit

percent

one can manipulate

9

is,

the

10

structure,

11

way.

manipulate

14

vector.

of

and then

its

very

the virus

those

the virus

This

16

read,

but

it

17

viral

vector

18

common

that

gives

vehicles

issue

in

form.

That

a plasmid-like

two factors,

we can then

and make it

you will there

that

an efficient

not

be able

Retroviruses

The most

adenoviruses,

23

advantage,

24

efficient,

25

expression

which

is

integrate for

long

are that so

terms,

it

often

used

will

there and that

to

are many different

one can use.

retroviruses,

that

and

associated viruses and Herpes viruses, and they ._ have pluses and minuses depending on the use that ..' will have for them.

22

a

is that

and change contents

easily

obviously

are

So

you have actually

in a circular

is to show that

ones

we

capacity.

important

and one can clone

15

that

room there.

SO given

13

One is

ITRs can be circularized

12

21

space,

us to use this

vectors.

105 percent

And the other

8

20

effective

wiggle.

7

19

have'allowed

enter will

one

as the

be

all

an cell

stable

one has no viral

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60

1

.. genes

in the most common of these And the disadvantages

2

to.produce. they

may integrate

advantages

randomly

often

sufficiently, the

9

into

the host

10

that

are

11

expresser;

12

vector

13

of by the host

14

In

are

that

so that

16

disadvantage.

useful

17

of

the it's

the

case

of

disadvantage.

advantages

what one does is put

22

the ElA/ElB

23

create

24

totally

25

vitro

region.

a little

So

the

for

all

integrate

a long-term

are

often

and it

gets

this

in

the

disposed

may not

be

other

vector

types,

depending

generation

the gene of interest

more space.

apparent

reasons

may be a

it

and disadvantages

The E3 region bit

not

vaccines,

what one needs to use them for. -A_ So typically on first

21

of therapeutic

quickly.

The same as for are

cells

the disadvantages

it's genes

the

enters

does not

immunogenic,

fairly

it

and thus

viral

and that

adenoviruses,

interest;

that

hard

cause mutation.

high expression

related

that

very

20

listed

chromosome,

often

15

19

for

they're size,

and thus

side

transgene

gene,

insert

flip

produces

8

there

is that

They have a limited

On the

18

vectors.

a

on

vectors instead

of

deleted

to

The E3 region

is

is often

irrelevant

for

in

expansion. NEAL R. GROSS

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61 1

as you can see,

But

more

of

the

3

vector,.and

4

leakage

5

transduction.

viral

genes

even though

there

So typically

7

the

8

wants,

9

In

that

and some expression

6

laboratory,

this

cells.

11

constitutively.

12

deficiency

13.

lot

These

14

express

gets

in the

used to infect

16

anymore virions

17

region.

18

In

that

19

approximately

20

mentioned,

21

product

22

often

23

milliliter.

24

through

a receptacle

25

receptor,

and it's

its

high

to

ten

to

It

affects

ElA/ElB

complement

cell,

will

of

virion

it

will

the a

vector, for

the

titers

in

the. 13th

viral

a variety.of

the

prevalent

ElA/ElB

virus

foreign

deficiency.

then

not make

not have this

has

DNA, as I One 1 can laboratory,

particles tissues.

per It goes

CAR for coxsackievirua fairly

cell.

or PER.CG the

once that

space

replication

them in very close

it

type

8 kb of

one

laboratory.

the target

because

in

and then one caa produce

And theoretically

15

this

a packaging

can entrance,

vector,

virions

is some

interest

293 cells

that

the

genes after

gene of

about

cells

of this

there

of those viral

talking

are

be a lot

within

is no ElA,

whatever

So they

of those

still

plasma to transfect

case we're

10

are

would

then one would create

clone

use that

there

adenovirus

throughout

nature,

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62 1

_ and then

it

2 3

tissues,

4

have high

also

uses some integrants.

And

it

such as antigen levels

5 evolutions

7

currently

8

is

9

That's

what

is

cells,

and it

been

many

What I've to

and it

described

as

the

that

second

12

had an additional

13

gene,

generation

first

generation.

can be E3 positive

or minus.

and again,

14 15

generation.

16

are

17

then the E3 region

18

various

farther

19

EIB with

a conditional

.

-only

I just ones

it

generation

again

generations

tissue

referred

ElA positive,

that's

they

in the E2 gene or E4

been quite

are

and then

or negative.

called

that

specific

either

E3 positive

They haven't

the

or so-called

is El minus as well, mutation

are

to you so far

The secondgenerationvectors

11

can

different

of adenoviralvectors

referred

El minus,

nonreplicating

expression.

have

of the types

10

21

presenting

there

in use.

into

get

of transgene

Now,

6

20

can

promoter types

X here.

and

or minus,

and

X.1 here so that

in which

They

ElB minus,

positive

like

to as any

is ElA and

they replicate

that

promoter

is

'active.

22

And the

23

is

24

closer

25

far

X.2,

which

are

final

generation

helper

to what a retroviral as

viral

gene

content.

at least

'dependent, vector

so far

and these

would be like Everything

has

are in as been

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63

1

~ deleted the

'2

from the vector

packaging

backbone,

sequence,

and it

except

the

ITRs in

has been replaced

by

some DNA content.

3

It does require

4 5

often

these

6

origin.

vectors

come with

I want

7 8

though.of

9

that

10

use.

11

in

12

comparing

to give

how this

critical

they it

were

to a first

14

the El and the E2 region

15

and the E2 region,

16

vector.

17

dose per kilogram

18

times

19

and this

At one time

it

is the platelet

et al., and

vector.

deleted,

deletions

light

bars

was ten

that

has

in the El

are just

to the

--

an El

these

ten to the 12th,

and one times count

be

their

generation

are a vector

-- one times

ten to the 12th,

for

bars

and the

some

examples

from O'Neill,

a second

So

may not

of vectors

generation

Here the dark

13

of

of changes

is data

using

DNA of

you a couple

generation

this

28 kb of DNA.

.stuffer

in the development

For example, which

at least

are three

ten to the 13th,

of the animals

or mice.

.* 20

As one can see,

the toxicity

21

j,-. ., slightly

different.

One times

22

but it

equilibrates

very quickly

23

the

12th

and one times So the

24 25

caused

by

this

ten

to the

at three

ten to the

12th times

dose, ten to

13th.

thrombocytopenia

virus

was perhaps

was no different

that

is in

often

a first

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64 1

generation

than

2

the

second generation.

And here

3

which

is

another

4

vectors,

5

llth,

6

times

ten

7

again,

at

8

difference.

is

again

using

known toxicity

of

and you can see that, one times to one

9

12th

is

times

ten

generation

11

as far

12

the

might

to

as the

give

a slight

toxicity,

but

this

adenoviral ten

then only

there

to

the

13th

the

a three

a difference,

So maybe the changes

10

enzymes,

one times

ten to the 12th,.and the

liver

and

there's

from first

no

to second

window of difference the

profile

in

seems to be

same.

13

Now, this

14

the

15

adenoviral

vectors,

16

generation

vector

17

is

generation

X.2,

a gutless

18

was not the case when we got to gutless

and here with

vector This

the

is

alpha-l

with

the

the

level

19

time.

20

generation

21

few days,

22

These are just

different

23

from 3.2 times

ten to the lith

24

10th.

These are weeks,

25

it's

or helper

what

we see is

anti-trypsin,

and this

of

expression

through

and you can see with

to baseline,

the first

within

the first

no expression

doses of the vector

And you can see

a first

same insert.

a peak of expression

leading

dependent

to 1.2 times

that

with

iater. ranging

ten to the

the

gutless

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1

vector,

2

this

3

viral

4

being.

5

immunogenicity

expression may be due to genes

in

much prolonged.

the

the

expressed

fact

that

first

generation

and

that

against

that

7

the

liver

8

peak.

9

vectors',

enzyme toxicity That

is

very

and it

are

tested

here with But

11

in

it

12

that

can induce

13

also

the

14

This

is

15

1999,

and what

,I6

color

is

a gutless

17

anti-trypsin

again,

18

first

is

the

that from

generation

that

20

animals

21

long term expression.

22

all

of

23

all

detectable

24

lost

the

received

first

the

only

the

adenoviral

in

et the

the

the gutless

generation expression

it

is-

response.

al.,

in

little

PNAS in

light

and it's

is

genes

In fact,

induce

This

This

doses

vector.

blue

human alpha-l

and in the multi-color

vector.

is

generation

any of

lines

is a

in baboons.

And what we see is that

19

This

first

at

Morall,

vector,

being

profile.

the

can

we see is

still

of genes.

immune response.

trans-.gene some data

not

are

and you can see a

occur

the gutless

genes,

still

content

clear

are

that

profile,

does not

Putatively,

there

And here is the toxicity

6

10

is

two of the veator

three

have a very

is out 100 weeks here, vector

animals

by 20 weeks.

and

had lost

Most of it

was

by ten weeks.

25

But there

was one animal

with

a gutless

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66

1

that

actually

lost

2

that

animal,

in fact,

3

to the

alpha-l

all

an immune response

6

addition

7

there's

variables

8

I will

just

9

this

10

rather

11

of

to

the

12

normally

vectors to the

variables for

that

very

to give

safety

all

of viral

couple

of

15

quantified,'

of

quality

16

that

17

know

18

functional

and can transduce

19

the

cells.

we can hetect those

target

how these

particles

vectors

with

this

is that

23

on how deep

this

soup

24

allowed

25

these

to progress, will

all

is,.

we also of

need to them

the gene of interest

these

reach

The

depending assay

variables, one of

a soup

cells.

the

are into

layer

particles,

how long

just

of particles

many

and many other never

to

are typically

on top of some target

problem

particles

but. then how

22

known

that's

I want

what we do is we just

of particles

on

agents.

easily,

Often

20

and

slides

control

and so we have the quantity

of

about,

not go through

to do that,

give

.fully

spoke

in

of the vectors,

I will

14

21

I just

brief

And in order you an idea

Of course,

you a sense of what is not

testing

done for

can be used to generate

the analyses

go very,

13

and

an immune response

coded.gene.

than what is known.

the

weeks,

anti-trypsin.

5

mostly

by ten

had developed

So these

4

etipression

gets

most of the

target

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67 SO may not

cells. detected

one time

be detected, or another

and whether is quite

So much so that

we sent

4

preparation

to six

5

differential

in

6

laboratories,

all

7

also

get handled

8

is just

a bit

9

et al.,

and you see here you can't

how they

10

but

there's

11

that

was

12

setting

13

vector

14

was not

the

shipped

just

So it

16

was the CO, that

17

there

18

in a very

I've

shown

turns

out in that

period

21

converted

22

They're

23

viruses.

24

that into

not

due

profiles.

Hoffman,

the distance, on, the

into

vector

a clinical only

from

and a vector

in,

particular

dropping that

that

the pH, and could

be lost

to

their

biology

gene

Additional

they

transfer

dangerous,

I think, can be

vehicles.

even as wild

type

have equivalent safety

of

the

second

NEAL R. GROSS (202) 234-4433

a

case it

adenoviruses,

Not alladenoviralvectors toxicity

This

of time.

efficient

inherently

and

ice.

So in conclusion,

20

by Neiber

town

tias a seven log differential

19

specific.

for .shipment

was seeping

short

those

adenoviruses

tell

on dry ice

on dry

from

differential across

was shipped

shipped

with

may be very

as an experiment that

titer

from a paper

log

an identical

and we had a two log

experienced

of data

get

variable. out

determined

a seven

15

25

laboratories,

they

COURT REPORTERS AND TRAN$CRIBERS 1323 RHODE ISLAND AVE., N.W. WASHINGTON, D.C. 20005-3701

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68

1 2

,.generation small,

vectors

temporal

may be transient

4

standardization

5

necessary,

6

of determining

7

entities.

8

a whole,

and I'm

11

standard

12

these

the

is very

there

a of

wild

things

as

a last

adenoviruses

there

is

developing so that

a

all

of

and quantified.

14

ACTING CHAIRMAN DADM:

I have. Thank

you

very

much. That

16

many questions

Stephens,

20

: ',

21

'regarding

was extremely

for Dr.

18 19

potency.

is

That's

17

of these

mention,

that

way

the data as

of clinical

can be compared all

is

standard

to analyze

group

type

specifications

13

15

a

because

and the quantity

assurance

working

clear

is a very

difficult

just

very

dose

the potency

And currently

actually

told

and thus,

9 10

is

of

It

in

stage.

And this

3

and only

Dr.

22

and retroviral

23

the

24

having

25

idea

status

Coffin

Katz.

difference

retrovirus

You said

something

between

adenoviral

that

an urban

been subject that

raises

and then Dr. Kohl and then Dr.

vectors of

It

us to consider.

DR. COFFIN: the

helpful.

I think

legend

to a real vectors

test,

on

vectors

has something

without

might

early

ever and that

of

actually is the

have some greater

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69 1

-danger

because

2

DNA into

3

don't

of

a cell,

the

property

in the cell

of

integrating

DNA, whereas

their

adenoviruses

do that. That's

4

actually

5

Adenoviruses

6

integrated

7

believe

that

8

numbers

are and I don't

9

what the numbers

do, of course, after

up

not

that

--

at

I don't

are,

know if but

entire

it's

10

make

11

integration

by

12

adenoviral

vectors

13

give,

14

of a fragment

15

a probability

of

retrovirus

16

administration

of

these

17

vehicles.

and in fact,

anybody quite in

they

21

standard

22

occasionally.

I didn't

mean to imply

23

more dangerous

or not.

I was just

24

of often

of

vectors

that

you

occasionally. operation,

high

after

or vaccine

It's but

they

as

is not

that their

do integrate that

reading

they off

were a list

events.

The one difference

25

doses

The fact

20

stated

the

as gene therapy

DR. AGUILAR-CORDOVA:

of

you in

DNA integration

19

method

that

DNA may be equally

Can you comment on that?

integrate

knows exactly

of the integration

18

do

the

efficiency

in

the probability

and I

what

likely

retroviral

of adenovirus

DNA does get

know exactly

difference

versus

course.

some low event,

difference

the

of

-- adenovirus

infection

the

true,

would

be that

they

do

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70 1

.no carry

2

at

3

integration

promoter

a

least~the

enhancer

promotion would

4

enhancement

But

COFFIN:

5

somewhere in the vector

6

a vector,

how

and often

9

comment.

10

is

11

yes,

they

data

or it

wouldn't

more than

compare I don't showing

and they

one,

So

,of

the

as

far

as

know that that

be

one

be any good as

Often,

if

yes,

integration

there

they

must

of course,

I

do integrate

--

and so can't

is any data.

There

on occasion,

persist.

12

ACTING

13

DR. KOHL:

Two questions.

14

Thanks

your

15

effect

there

DR. AGUILAR-CORDOVA:

8

in the LTR.

be different.

DR.

7

in there,

Dr.

CHAIRMAN DAUM:

for

talk.

It

Kohl,

please.

was enjoyable

and elucidating. You mentioned

16 17

Could you elaborate

18

get

19

deleted

20

into

enough

the

21

leakage

that

that

the

concept

a little

from

you can get

further,

a gene

that

a competent

leakage.

and can you supposedly virus

is

injected

host? DR. AGUILAR-CORDOVA:

22

I was speaking

23

deleted.

24

on there.

25

of

about

Obviously

However,

So the leakage

was from the genes that the one that's

there

is

deleted

the

that

were not can't

potential

leak

for

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71

1

- recombination

with

leading

the gene inside

2

and thus

3

believe

that

4

PER-C6

cells

5

between

them and the 293 cells..

to a replication

that

will

were

7

more on the toxicities

8

causes

9

The platelets,

10

other

that?

developed,

and

the mechanism

the

liver

function

know are what the toxicities

13

course,

14

University

15

to

16

injected

as you've

heard,

dose

into

of

their

there

19

have seen in many animal

20

enzyme content,

21

not

22

seen the

with

upper

be

25

leakage

the

toxicity? and

animal

So what

we

and particularly,

was an incident

of in'the

vector

due

directly

artery.

respiratory

often

distress,

models

transient

models.

and what we

is an elevated

liver

and recoverable,

and

Many Phase 1 studies

have

same thing. Thrombocytopenia,

to

What

case what was seen was a DIC like

syndrome

24

bit

where a young man died

hepatic

18

23

a little

Yes.

an adenoviral

In that

just

are,

of Pennsylvania

17

difference

as well?

12

a large

of

I

transmission

DR. AGUILAR-CORDOVA:

11

virus.

as why the

the

Can you elaborate

cell

of the adenoviralvector?

What's

toxicities

competent

be spoken to as far

DR. KOHL:

6

the packaging

caused because

due it

to

believed

endothelial

is consumptive

cell

but

not

shown

damage

and

thrombocytopenia.

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72 1

DR. KOHL:

2

DR. AGUILAR-CORDOVA:

3

probably

4

potentially

5

infection.

cytopathic

effects

ACTING Stephens,

Dr.

Katz,

10

gene therapy

vectors

11

like

12

would.

13

versus

the E3 sequence

15

in or out

of clarify

in this

discussion

18

gene therapy.

19

had

20

accepted

'21

applications

22

vaccines.

that

discussion not,

23

and

trying

And Dr.

24

E3 region

25

he's

big

in

early

prop.onent

for

us,

if

you

lot, many in

Sure.

too, of

So not

be

just

of vaccine the

the

cancer

started

I believe,

or

RAC we've

what

to vaccinate

of leaving

should

vector.

a confusion

Ginsberg '8Os,

of

and I'd

that

as a member of a

there's

vectors,

you think

delivery

especially We're

Dr.

the questionrelatesto

is there I think

or

have

the issues

issue

DR. AGUILAR-CORDOVA:

17

I

about

that

and whether

of the vaccine

16

and

to the original

discussion,

vaccine

More specifically,

14

is

Dr. van der Eb.

in my mind confusion

you to kind

vector

CHAIRMAN DAUM:

some at least

9

the

response

DR. STEPHENS: In this

8

The mechanism

of

an immunological

6 7

Who does the mechanism?

should

gene are,

be

therapy in

fact,

against

cancer.

working

with

or before,

the E3 region

an and

in when

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73 1

..

one wants to create

2

situation,

3

is

4

immune response

it

an immune response

is possible

an adjuvant

5

to

whatever

The flip in my laboratory

7

a gene

that

8

against,

for

9

tend to get an awful

one

of

t.hat

to

with lot

ACTING CHAIRMAN DAUM:

13

Dr.

14

DR.

and there

18

a module

19

and its

21

in

could

some

vector,

we

adenowirus

and

go either

way. Thank you.

please.

KATZ :

What

and what cells

is

the

express

receptor

for

the receptor?

DR. AGUILAR-CORDOVA: The known receptor,

17

20

that

an

the gene of interest.

12

16

create

a CTL response

of CTL against

So it

adenovirus

to

an adenoviral

11

15

itself

when transducing

create

to none against

Katz,

is

and others,

wants

example,

little

the adenovirus

one wants

side

studies

very

in that

against.

6

10

that

because

'7 cells L some

are, probably called

the known receptor

CAR, coxsackie

distribution

is fairly

are especially integrants

others,

adenovirus ubiquitous.

high expressers,

ACTING CHAIRMAN DAUM:

23

DR. VAN DER EB: the

25

Even a deleted

issue

of

receptor, Epithelial

and it

also uses

as co-receptors.

22

24

is

leakage,

I'd

leakage

or undeleted

Dr.

van der Eb.

like

to come back to

that

you

vectors

mentioned.

are supposed

not

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74 1

_ to express

2

present

3

is

4

the

the

the rest

of the viral

in the vector. master

rest

switch

that

of the viral

6

time that

7

of

8

multiplicities

the

is because

directs

known already

leakage

may occur

viral

genome

For

10

ElA-like

11

of

the

rest

12

reaction

of

13

the host.

reason

activity

14

the

the

expression

host

for

occurs

a rather

for

efficient

16

region

then

when

this

very

creates

and leads

viral

genome

cell

17

some gene expression,

18

levels

to produce

19

although

But given

20

1-expression

21

absence.

of

the

consequent reaction

from

long

genes,

seems like

the El

there

is

maybe not at sufficient

the data, other

of

So in order

and to create

DR. VAN DER EB:

22

it

of

to expression

with

of the other

However,

virions

high

a kind

immunological

transcription

is necessary.

long

the rest

DR. AGUILAR-CORDOVA: Right.

15

of

are used.

in the cell of

the EIA gene

and expressionof

of infection

9

the

is still

genome.

Now, it's

5

23

That

genome that

there

viral

the El.

seems to be some

genes

even

in

its

Is the data of Tom Shenk

ago?

24

DR. AGUILAR-CORDOVA:

25

ACTING CHAIRMANDAUM: Two last

Right. questions,

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75 1

~ please.

2

DR. COOK:

3

question

about

4

point

5

travel

6

theoretically

7

trying

8

that

9

it

of view to

the

I'd

E3 in or out. is

to shut

surface if

that

to respond

to the

E3's job from the virus'

down Class Golgi

were uniformly and it

in the cell

1 expression

or

mechanisms.

So

true

required

and you were expression

of

in which E3 was co-expressed,

would be a good idea maybe not to have E3 present.

10

The

truth

11

adenovirus

12

express

13

infection.

It

14

hours

infection

in normal

after

is

doesn't

that

are

could

17

the

18

greater.

19

system,

20

consider

21

expression

22

antigen

presentation

23

surface

of the gene of interest.

24

So it

ElA,

might

on the

very probably

is

surface or

in

48 to'72

expression are.

E3 effect on how. you

at least,

E3

don't

late

peptide

the

depend

but theoretically, whether

that

on what the kinetics

co-expresses

with

virus.

the

16

cell

is

happen until with

depending

YOU infect or cells

phenomenon

So chances occur,

when

human cells

that

ElA,

15

25

like

through

to make a vaccine peptide

just

is

much

rate

the

one would have to

downregulating and whether peptide

If

Class that

expression

ACTING CHAIRMAN DAUM:

Thank

1

alters on the

you,

Dr.

Cook. NEAL R. GROSS. (202) 234-4433

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76 1

DR. BLAIR:

2 3

retroviral

4

there's

5

the virus

Yeah.

background,

as it's

This

but is there

an encapsidation

6

of

cell

comes out

of my

any evidence

that

DNA/RNA protein

Not

at

8 I-

certainly

9

proteins

the

level with

that the

we've

RNAs,

or viral-like

10 that,

12

recombination

13

events

and

certainly

that

14

much, Dr.

scheduled

18

We'll

19

der Eb.

break

reassemble

the possibility the

of

recombination

random pieces

we

will

of DNA.

Thank

shorten

to a 15-minute at

lo:55

you

very

the record

at IO:39

a.m.

22

the record

at lo:58

a.m.)

23

ACTING CHAIR.P@N DAUM:

the foregoing

down as quickly

20-minute

I have 10:40.

and continue

21

settle

the

break.

(Whereupon,

25

of

possibility

non-specific

20

please

viral-like

Aguilar-Cordova. And

17

retroviruses

and so on.

there's

package

I know of.

of

ACTING CHAIRMAN DAUM:

16

24

in

especially

there's

events, would

seen

particles

But certainly

11

in

assembled?

DR. AGUILAR-CORDOVA: Not that

7

15

Blair.

Dr.

..

with

matter

Dr.

van

went off

and went back on

as they

ACTING CHAIRMAN DAUM:

Would

everybody

can? We're

ready

to

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1

continue

with

the open session. We will

2 3

tell

4

and the

5

manufacture

now call

us about adenovirus development

transformation

of defective

adenovirus

Welcome,

7

DR. VAN DER EB:

8

So what I would to

of human cells

of 293 and PER.CG cells

6

9

on Dr. Alex van der Eb to

for

the

vaccines.

Dr. van der Eb. Thank you. like

to do is to describe

you how and why we have made two different

10

lines,

11

which

12

made in

13

material,

14

Leiden.

15

from human embryonic

16

fetal

tissue

17

that,

so that

18

the PER.CG cell

19

in 1995 from an embryonic

20

made from fetal

21

1985.

adenovirus are called

transformed

and

also

was prepared The 293 cell

the

cells,

by myself

kidney

were

starting

at the University

cells

that

of

were made from one year

was in 19 -- probably

before

in 1972,

whereas

was made by Ron Bout and Frits retina

cultures

tissue

by me ten years

just

shows you again

genome and you have seen it

24

this

25

transform

was due to the cells

lines

the

ago by myself

23

virus

Both cell

lines

was made by Frank Graham in 1973

one year

This

22

human embryo cell

293 and PER.CG.

my lab,

cell

in tissue

already. fact

that

before

were

that,

in

the adenovirus The interest

that

culture.

Fallaux

the In fact,

viruses all

in can human

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78

adenoviruses tissue

or

almost

culture,

adenoviruses

and can

all

can transform

cells

in

that

certain

types

of

tumors

in

experimental

also

induce

animals. The transforming 6

survived the

8

-- the transforming

left-most

harbors

about

percent

the

with

genome that

could

I

human cells will

tell

human cells,

possible

virus

with

you

and it

in the first viral

we got

started

my lab

the

actually

developed

the

technique,

place

which

to make infectious

DNA.

you transfect

the

Adenovirus

Type 5 into

permissive

infectious

virus,

it

a pointer

all

Graham in

-- in

be transformed, how

DNA transfection

intact If

have

of

whether

phosphate

made it

18

is associated

in transforming

1972 when Frank

calcium

16

region

I hope it

We became interested

therefore,

transformed in

ten

-- oh,

the El region.

the question and

region

but here?

also

There

intact

viral

DNA of

human cells

you get

turned is

out

--

do you

no pointer?

Okay.

Thank you. And it 23

possible

24

cells proved

turns

to get infectious

with

the intact

capable

out virus

viral

of transforming

that

not

only

bytransfecting

was human

DNA, but also purified cultured

it

rodent

DNA cells,

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79 1

.but

human cells

could

not be transformed.

And the reason

2 3

got

destroyed

4

If

5

Daltons,

6

that

7

remained

8

viral

9

genome is necessary

the

by the

viralytic

DNA was sheared, up to

three

million

Daltons,

potential

a rather

As

I

itit0

small

for

purified

13

transformed,

everywhere

interested

14

human cells

by adenoviruses

just

15

whether

permissive

18

that

19

transformed

20

smaller

21

cells.

human

could

be perhaps

done

if

pieces,

24

there

25

cloning

with

cells

cells

yields

could in

not

be

transforming

in order

to find

rodent

detailed

out

time;

that

could

was sheared hamster

activity,

be into.

kidney

and this

studies

enzymes.

permissive

from the fact

cultures

were Syrian

shearing

were no restriction at that

cell

the DNA of adenovirus and these

that

transformed

The transforming

23

5 DNA

is possible.

semi-permissive

22

of the

of the viral

human

But we found some evidence human cells

out still

Adenovirus

effective

17.

cells

proportion

12

16

turned

a portion

transfected

that

it

transformation.

said,

but

reaction.

of rodent

11

virus,

human cells

up to 3 mega delta

indicating that-only

genome,

10

these

(phonetic)

however,

the transforming intact,

is that

at There

the transforming

that

was time;

was no DNA activity

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80 1

. associated

with

the

2

percent

3

the basis

of the

4

humancells

with

of the adenovirus

6

human cells

7

human cells human

is

answer

can be transformed

adenovirus,

and

DNA is

10

that

same

11

transformation

12

more?

the

if

required area

the

which

to

transform is

cell

or is

transformation

of human cells?

15

that

time

because

16

evidence

17

with

cancer

18

open

question,

19

still,

20

human adenoviruses

21

men.

and

although

fact,,

is is

have anything

23

human embryonic

24

And that

is mainly

25

system,

the rodent

method

kidney

that

because

Is

needed

for

less

or is

it

a model to

And that

was at

there

was no

at

to do

this

we followed Why kidney that

an

moment

no evidence

of the fact

model that

cells?

to do with

cultures.

the

an open issue,

it

there

of

have anything

was still

clearly

So the

22

in

it

it

although

human adenoviruses

whether

part

develop

study

in humans,

to transform

also

14

that

of the

by adenoviruses,

so,

And then can we simply

important

affect

question

at all

that

of rodent

13

all

by adenovirus

why we wanted

to

11

of adenoviruses.

reason

just

left-most

genome, and this

fragments

adenovirus

9

percent,

transformation

So the

5

.8

11 left

that

cancer

in

was take cultures? the rodent

we used were always

baby

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81 1

.- rat

kidney

or

2

kidney.

3

transformation

baby

The kidney

kidney

kidney

cultures

with

6

with

7

DNA of

8

enzyme fragments.

the

with

calcium salmon

sperms.

10

available

11

293 cell

12

simply

13

the

was made with

rodent

for

with

17

probably.

sheared

transformed

was as follows.

an unknown

family

20

for

normal.

the abortion

it

at

usable.

colonies

enzyme

and also

Adenovirus

that

material,

history, date

the

5 DNA, then as we did with

Nothing

the

it

in 1972

as I can remember

got

was,

known anymore.

was wrong.

but

kidney

of the fetus

were unknown to me. time,

fetal

was obtained

is not

as far

The fetus,

18

completely

restriction

not yet

The kidney

The precise

19

carrier

we had resection

So the kidney

16

5 DNA

cultures.

14

material

DNA.

using

was not

They were just

these

Adenovirus

technique

later

for

human embryonic

to make pure DNA, but the first

scored

15

these

So this

hamster

adenovirus

purified

phosphate

baby

suitable

sheared

sheared

One year

9

or

were very

When we transfected

5

22

cells

studies

4

21

mouse

was

The reasons I probably

knew

lost,

all

this

fetus

were

then

information. The

23 24

isolated

25

called

kidneys

and the kidney still

air

cabinet.

of cells

the

were isolated

in the SO-

There were no laminar

flow

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82 1

.-hoods

at

that

time,

2

cabinet

3

and worked quite

4

to sterilize

that

and this,

was also used all well.

it,

cells,

7

the

simply

over for

a still tissue

There was W lights

and that

air

culture

in it

just

was all.

So as we did

5 6

is

also.

for

the

rat

kidney

surrounding

membranes

were

removed

completely

as possible,

and the

kidneys

were

8

minced with

scissors,

9

were

recovered

10

cultured

11

serum.

in

trypsinized,

after medium

That

containing

13

a commercial

14

else,

15

blood,

calif

cultures

18

time.

19

all

20

-being ..

that

the

were

trypsin

bovine

We either lab,

serum,

calf

got, it

not

from

or we made it

from

somebody

ourselves

from

blood. Rodent,

17

and the cells

serum was obtained

caif

source.

from another

16

then

is what we know. And this

12

removing

as

took So there

monkey,

place

and

in the

same general

was one cell

of the experiments,

other

all

human area

culture

room,

the cell

'culture

cell

at that

and there work was

done. There

21 22

but that

23

used in addition

24

the

25

possibly

was also

was in a separate

oncogenic also

experiments virus

to Adenovirus Adenovirus already

12,

Herpes

'with

viruses,

cultured

unit,

and we

5 whole

viruses,

also

as well virus,

as

SV40 and

but maybe Herpes

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83 1

_ virus

was not yet

2

So the

3

Adenovirus

4

prepare

5

virions,

6

in

7

eight

8

at that

9

as I already

10

used at that method

5, was isolated the

time:

was DNA from from virions.

DNA by first

growing

and the DNA was then

this

case

through

million

calcium

and purifying

fragmented

and the DNA fragments with

the

by shearing up to

There was no cloning

indicated

type

So we had to

a 22 gauge needle

Daltons.

time,

wild

about

strategy

were transfected

salmon sperm DNA with

the

technique.

11

The results

12

the first

experiment

13

were not a single

14

it.

Again,

15 16

found finally

17

in

18

after

19

transfection. .--

were rather

of quite

disappointing.

In

a number of dishes

there.

transformed

colony.

no transformed

colony.

However,

many other

after

one transformed

So we repeated

experiments,

colonywhichwas

we

visible

i

20 21

the

cultures,

and that

transfection

was

This colony,

and established

23

and that

24

seen after

25

cultures

appeared

seen,

33.

single

and became the There

22

this

colony

is because the cells were fixed

were one,

days

colony

after

was picked

293 cell.

two colonies the

33 days

second

here

mentioned,

colony,

was only

at the end of the experiment, and stained,

and one other

the colony

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84 1

was seen at the edge of a dish

2

So the

3

would

give

4

The cells

5

culture,

6

ampules.

rise

grew at all,

was possible Only three

7 8

faster,

9

time

but

still

11

human

cells

12

Adenovirus

13

extremely

14

cannot

15

transforms

5. well

rodent

these

why human cells

18

adenoviruses.

19

came out

20

mutation

21

transformation.

are

with

the

quite

so

line

that

it

Another

22

they

same DNA that

also

it's

still

kidney

24

course,

of many different

cell

25

one

that

unclear

transformation

is

that

had

few cells

293 cell of

permissive

which types,

the

by

some kind

is that

human embryonic

very

replicate 5,

became

possibility

are

that by

to

that

primary

there

a week.

cells

moment,

23

--

and a doubling

efficiently.

resistant

a cell

to grow

Adenovirus

One possibility of

started

transformation

replicating

cells

was that.

from these experiments

Although

So up to this

17

four

poorly

to

be transformed

16

passage

resistant

in

months in

down the number of

a week or more than

are

which

to expand. five

time the cells

So it appeared

10

difficult

to free.ze

relatively

was at least

colony

and after

ampules,

And at that

we had missed.

transformed

to 293 was very

hardly it

single

which

since

to

this

consists, that in

is of

there the

a

is

whole

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85 1

T culture

which

2

that

3

transformation

this

particular

possibility

6

cell

7

and this

8

traveling

9

possibility

in that

something here ,to that

10

cells

appear

11

adenovirus.

12 13

fibroblasts

14

result.

15

cells.

to

that

also

for

transformation,

tried

have

and

around

fibroblasts,

that

page

example. crisis

time,

the

22

gradually

started

23

you

to

24

Nothing

skin

any positive

cells

the There

And then

the

13,

that

lung

the

cells

went

is also

seen

in human diploid

They always lasted

to die,

defeat

happens.

by

human embryonic

is followed

This During

neural

diploid

never

a

transformation.

19

21

'is

transformation

the same type of crisis

20

so that

human

tried

know,

to me when I was

to

when SP40 transformation

25

We don't

occurred

one

a neural

be more prone

18

have

is actually

culture.

We

for

those

but

testedbecause

Anyway, crisis,

and of

probably,

Gaithersburg,

No positive

into

one

canprobablybe

We also

16

know

the 29? cell

was present is

came from

never

is that

that

transformation,

cells.

We will

5

to

cell

prone

4

'17

are permissive

go in crisis.

nearly

remained

three on the

months. dish

some of them at least. cultures is no cell culture

for

a long

or So

time.

division. started

to

recover

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1

for

some reason

2

case of SV40, but apparently

3

begin

4

what happens

to

not in the same way as usually

grow,

whatever

in this

that

crisis

After

5

activated,

,7

the

8

increased

9

then

by Frank

10

went

to Anestilles

11

have been published

were

when

the adenovirus

14

not

15

and this

16

also

17

not expressed,

the

in several

papers.

sharp,

some E4 region

but

adenovirus

DNA also

20

became interesting

21

this

22

about

gene

23

first

retroviral

24

vectors.

occurred

1974,

where he

and the

it

doesn't

really

in these

So this

is

matter, There is

cells,

to

basic

became interesting,

which

is

for

introducing

research, Adenoviruses

gene therapy.

in the '80s when people

And adenoviral

It

of

is in the 293 cells.

addition

vectors,

data

the part

in the 293 cells.

as factors

therapy,

rate

were shipped

4,041 nucleotides.

however.

i9

growth

to show here also

present

in

recovering,

in Canada,

in

genome present

Now,

is

and

(phonetic)

is the left-most

18

telomerase start

Graham to McMaster

completely

know

Severalambules

I would like

13

We don't

cells

subcultured

significantly.

12

over the plate

base.

apparentlywhenthe

cells

all

means.

crisis,

6

25

cells

in the

started

genes

'and also

later

vectors,

in

So

to think

into

cells,

adenoviral

contrast

to

NEAL R. GROSS (202) 234-433

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a7 1

._ retroviral

vectors,

2

said

today

3

of

4

and the reason

5

was,

in fact,

6

293

cells,

7

packaging

8

vectors

the

in fact,

a deletion

El gene,

have as have been already

in the El gene and in the place

you can clone that

El deleted

the present which

cell

so

9

being

used

were

11

transfer

purposes.

12

it,

13

certain

disadvantages

14

vectors,

for

15

of important

to

more

and

also

The

more

for

compared

gene

you've

reasons.

heard

They have

to

but they certainly

cells

to

deleted

grow

retroviral

have a number

adenovirus

the

replication

vectors

were

deficient

El

18

available,

and those

were the 293 cells,

19

in

1994 the

first

20

already ._ deleted

21

Crystal

in '94,

22

correctly,

23

trial

adenovirus

clinical

vector

which

was

probably

the CFTR gene.

So there Two, nine,

study done,

was the,

with

was if

for

an El

made by

I remember clinical

vector.

was a new use now for

three

also

and in fact,

That was the first

gene in an adenovirus

cells.

viral

adenovirus

vectors,

similar

suitable

El genes.

17

25

very

advantages.

16

24

of the

recombinant

for

example,

was chosen

generation

the

Adenovirus

suitable

be

first

expressed deleted

vectors

are quite

these

El

10

interest,

adenovirus

out

for

they

gene of

or the availability

turned

line

because

the

quite

some period

the

293

of time

NEAL R. GROSS (202) 2344433

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1

'. was the only

packaging

2

of adenovirus

vectors.

3

deleted

El

4

became common practice.

5

to use adenoviruses

6

limitations

7

It

became apparent

a

to

recombination

9

cells

into

10

gave

rise

11

adenovirus,

12

difficult

13

and of

14

vector

15

be physically

at that

RCA,

with

and

the ideal

ia

we decided

19

you here,

sequences

of

replication

turned

around here

that

the

identical

used,

this

and instead

be

very

vector, to

the

cannot

really

and therefore,

first,

just

vector

in which

of

the

it,

to show the

gene of

here. are

23

proportion

24

the El gene and P9, protein

25

be quite

293

the

adenovirus

a considerable

to

293 was not

is the recombinant

can be inserted

of

competent

from the vector.

19 -- oh,

here

293

and this

and therefore

gene therapy

deleted,

due

the

of RCA free

almost

293.

is that

occur,

out

batches

was clear for

line,

from

could

it

started were also

cell

vector

RCA is

separated

And

22

El

growth

transfer

there

and that

the gene of interest,

vector

is

but

time,

large

the

So it

for

gene

packaging

formation

course,

for

as a factor;

the El deleted

17

21

vectors

between

to

available

More and more groups

to produce

-El gene ', interest

line

to the available

16

20

cell

cells

with

genome integrated 9, and there

overlap

at both

turns sides

a with

out to between

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89 1

the

recombinant

2

vectors

of both

sides

vector,

which.is

essentially,

again,

So the

5 6

vector

yields

7

replicating.

8

concentrations

9

don't

10

dissemination

11

also

12

together

of

type

This

it.

It not

the it

14

virus

strains

15

where

the

16

example,

17

other

18

replication

19

partially

only

could

that

replicates

theoretically

also

in

can. attach So in

that

that

case of multiplydelete'dvectors,

23

that

24

E2A,

25

vector,

will

can

could

22

El

still

yield

modified such

other

new vector

a way,

for

receptors

when say

that

that

yield,

and

becomes

this

give

containing

to deletion

example,

but

is

a'

you have created.

replication

in addition

to case

you

21

it

virus,

factor

uncontrolled

same cell.

competent

for

type

modified

deficient

you

wild

is

it

where

the

capsid

Also,

high

to

capsid

new virus

toxicity,

of

rise

case of

it

capable

in places

the

cells.

20

cause

in

that

is

give

of

in the

It

which

of virus

could

type adenovirus.

of the El gene in the

virus,

recombinant

with

13

the wild

could

perhaps

want

generation

you can get back RCA,

combination

wild

first

of the El region.

And be recombination

3 4

adenovirus

when El

for

viruses example,

of El also is

to

in

the

vectors

deleted

reinserted

be replication

rise

deficient

in E2, into

the

because

NEAL R. GROSS (202) 234-4433

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90 1

the E2A gene is absent,

2

immortalizing

3

you have created. So in 1995,

5

Fallaux,

6

from our university

7

we should

8

matching

factor

9

sequence

overlap

Brahm Bout

try

sequences

Gene Therapy

such

cell

that

allowed

13

vectors,

14

pharmaceutical

15

you can just

16

the

17

Adenovirus

of

as well for

the

in order

RCA.

cells

20

because

21

transformation

22

worthwhile

that

time.

these

cells

it

that

line

there

and is

no

and the advanced

try

to make a new system

It

of adenovirus

should

to do that,

also all

over again

and it

of

meet

could

multiply

be

deleted

also. the

human embryonic

Why not

kidney

we didn't

retina

cells?

Simply

to adenovirus

think

it

would

be

all.

Human embryonic

23

that

were so resistant

that to try

cell

If you start

So we choose

19

Fallaux

decided

production

manufacture

5 vectors

that

line.

standards.

ia

Group,

is

Frits

and Frits

factor

pharmaceutical

three

basis

the

virus

(phonetic),

a way

between

And indeed,

12

Brahm Bout

from IntroGene

in

in theory,

competent

and make a new helper

in the

11

at

vector,

or transformation

4

10

but this

24

Gallimore

had shown not

25

embryonic

retina

long

retina

was chosen because

before

was permissive

to

that

that

human

transformation,

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1

.- could

be transformed

2

12,

3

in

4

embryonic

and that

was,

animals,

7

studied,

8

transformed

9

a real

10

based on some other we decided

to

5 and studies

take

human

cells. can be transformed

at least

in some of the cases that

there

is no real

crisis.

by Adenovirus we have

So the cells

become

and then go on to become immortal

crisis

in

which

the

whole

culture

without stops

the

fighting. Transformation

il 12

efficiency,

13

it

is

but

is

anyway,

there

So I isolated

15

healthy

16

old.

17

or the

pregnancy

18

weeks,

and it

19

abortus',

20

because

fetus

as far

turned

abortus

23

before

October

out

rather

low and

from a'fetus, be seen, with

of

provocatus,

normal

and

to get

this.

rid

There

from a 18 weeks

a family

history

up to

to be a socially

was, however,

that of the

the

indicated was

simply

fetus.

was permission,

was in 1985,

18

et

ten years

this. This

24

special

the woman wanted

and that

retina

was completely

We got cetera,

a

is transformation,

as could

There was nothing

21 22

still

reproducible.

14

25

Adenovirus

So they

5

5, and also

again,

and therefore, retina

6

by adenoviruses,

shows that

I as, Leiden

University

the cells

were isolatedin

in my lab.

They were

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92 1

then

isolated

inseparate

2

contained

3

did it

4

cell

5

That was only

6

cell

a laminar

air

in the cell culture

cell flow

culture

rooms that devoted

culture

cabinet,

area, and that

area of the three we had available

to diploid

cell

which was we

different

at that-time. cultures,

human

cultures. The cell

7 8

from certified

9

should

10

nitrogen,

11

for

say,

supplies. the

construct,

14

between

15

allow

16

production.

were frozen,'

of the defined

in

to

order cells

18

regulated

not

19

promoter,

and the

20

functionally

21

interested

genes

by the

23

University.

There

24

was

The

25

different

this.

colonies

and

El

the

thing

El

but

was all

that

PGK

sequenced

and.

out

at

DNA used. yielded

about

18 days,

COURT REPORTERS AND TRANSCRIBERS 1323 RHODE ISLAND AVE., N.W. WASHINGTON, DC. 200053701

were

by the

of the viral

was no carrier transfection

would vector

I can show you if

was carried

DNA

homology

deleted

NEAL R. GROSS (202) 234-4433

liquid

PER.CG cells

on expression

after

I

was thawed

sequence

vectors,

in

characterized. the data

in

files

ElA promoter,

whole

'85,

identifiers

free

Transfection

22

El,

and the

The El

course,

already

stored

eliminate

RCA contamination

17

of

PER.CG cells.

We used

the

time

1995 one of these

the generation

13

media were,

At that

cells

and in

12

culture

you are genes. Leiden In

'95

a number

of

and one of

i www.nealrgross.com

93 1

several

of those

were isolated.

2

finally

was established

3

Clone

4

of viruses

5

and ElB gene products.

6 was chosen

and gave rise

because

and also

7

it

is possible

8

I remember

9

sometimes

that

from

10

university

12

picked,

13

was close

14

building,

15

by them.

in

yield

had rather

high

expression

of ElA

did not go through

in some case a crisis

crisis

and

in

materials

18

down at IntroGene,

were used,

after

Leiden,

shows

20

recombinant

identifiers

21

gene is still

22

of

is

the

23

the gene of interest

is

present

and here

in

cell

gene construct

colonies

were

to IntroGene,

which

fact,

the

control

same

was done

area defined

banks were laid

29, 33, and 36.

you

the

itself

it

area

in

culture

information.

The

shown here.

at the right

with

that

at the

Cell

hand and,

where

El is

The P9 in fact,

deleted

and

inserted.

Then the PER.CG cell El

as

event

the

of course. passage

This

J-9

24

Gallimore,

and the whole documentation

17

but

may be observed.

In the dedicated

El,

Phil

was transferred

by also

16

25

of

crisis, appears,

the transformation

Leiden

everything

to PER.CG, and

the highest

So after

11

6,

gave

experiments

a short

Clone

it

These cells

6

One of them,

was transformed

a PGK promoter

by an

and a polyacid

NEAL R. GROSS (202) 234-4433

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94 1

.. of Hepatitis

B virus,

I believe.

And here is no sequence

2 3

RCA has never

4

experiments.

been observed

5

And this

6

you some comparisons

7

I remind

8

for

you that

different

in many,

is the

final

between

both

cell

The objective,

10

-- was basic

11

transformation

studies

12

studies,

but

gene

13

embryonic

kidney

14

to now,

research,

many different

slide

just

showing,

293 and PER.CG. lines

Again,

were made in my lab

as I indicated,

after

that,

expression

studies

in the years

manufacturing

of adenovirus

17

PER.CG is RCA free.

20

done

21

information

22

this

is

23

time

was a little

24

in a laminar

human

following

that

up

time

pharmaceutical As to RCA free,

three

is not.

documentation

out completely

that

for

vectors.

Two, nine,

The history has been carried

with

say.

16

19

293

not transformation

PER.CG was made just

18

is for

and we have done many different

cells

I would

15

25

and real

reasons.

9

at

homology

for

for

293.

of the cell

PER.CG and was not We -had

on 293 or what was available available

for

PER.CG.

primitive

flow

no

got lost,

Containment

perhaps

line

at

donor and that

and was now done

cabinet.

The serum sources

were of

noncommercial

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95 1

.. use.

Probably

2

samples

3

medium,

--

I

have

supplies

made it

were

Crisis

5

had a crisis,'

6

and these

free

history

was not crisis

cells,

for

serum

and

just

means that

293

free

at the long

PER.CG had no crisis

for

crisis,

some reason.

Andthenpharmaceuticalindustrystandard.

7 8

I

9

PER.CG were made for

realize

that

10

as far

11

have taken

this

as I know, license

12

sounds that

characterized,

14

is

a bit

for

commercial,

particular

more than

purpose.

50 different

PER.CG.

Two, nine,

13

three

but Also,

companies

1

was not

is in the public

in the

domain,

same way

whereas

PER.CG

licensed. So I

15

think

I'm

16

wants to see the data again

17

on,

but

I don't

18

think

at

the

of virus

that's

end if

much, Dr.

very

then

22

take

a couple

Ms. Fisher. DR. DECKER:

Dr.

important. Thank

of

you

questions.

very

is not capable

human diploid

24

of cytolysis?

The human cell DR. VAN DER EB:

Dr.

Kohl.

Did you say that

23

25

and so

van der Eb. We'll

Decker,

somebody

production

ACTING CHAIRMAN DAUM:

20 21

now used

Certified

et cetera.

4

19

myself.

cell

transformer

adenovirus because

-Yeah,

yeah.

Well,

yes.

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96 1

What I said

2

a virus

is

you --

and you put

types

that

4

skin,

embryonic

5

and that

'3

that

DR.

8

adapting

9

might

wipe out a whole

DECKER:

Does

and human diploid it

that

11

possibility.

12

different

13

tested

14

DNA, also

15

of the DNA.

16

that

I don't types

were

of diploid

by fragments

that

17

tissues,

19

be transformed,

20

be that

cells

later

--

23

neural

of neural

24

this

25

is that

that

is a

the

that

three

we have just

by

fragments

but I don't

believe

there

might

be other

in the human body that retina

known that

more clearly

five

because

also,

are also

22

cells

it

transformation

example,

cells is

cytolysis

issue.

for

It

21

an then

that

human cells

and tissues

neural

the

Theoretically

to

What I think

18

might

of the DNA, resection

We did it

is a big

that

capability?

believe

so resistent

area.

imply

get

the

reaction,

adapted

way so you didn't

the

culture

that

cell

DR. VAN DER EB:

10

DNA or

kidney,

Then you see lytic

unmask a transformation

is

intact

on human diploid.cells,

lung. just

6

attenuated

you take

we have used is the embryonic

will

7

it

if

cells.

It

could

Type

12 is

transformed. Adenovirus

much more efficient origin

can

in

than

we are talking

Adenovirus

about

ACTING CHAIRMAN DAUM:

transforming 5, but

here.

Thank you.

NEAL R. GROSS (202) 2344433

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97 1

Ms. Fisher,

then

2

MS. FISHER:

From your chart

3

there

was no cell

crisis

with

4

DR. VAN DER EB:

5

MS. FISHER:

6

that

you said

DR.

7 8

believe,

9

in

slowed

11

did

type but

not

he just bit,

14

the whole

know.

culture

just

Minor,

There

took

took

the

off

DR. KOHL:

1‘8

prion

19

specifically

20

especially

transmittable about

off

cells

again. that

be a crisis,

again

the

is a but

and continued. then Dr.

like

fetal

the

possibility

can you calf

tell

of

us more

history

of PER.CG,

certified

sources,

'85?

You said I'd

Regarding

diseases,

back in

21

but

I

one or two weeks

not say that could

been,

by Gallimore that

and during

That

have

observed

described

observed.

please.

17

it

was from

--

23

DR. VAN DER EB:

24

DR. KOHL:

25

crisis

ACTING CHAIRMAN DAUM: Dr. Kohl,

15

that

--

was a short

of crisis

I don't

you said

-- use .of PER.CG, but before

So you can perhaps crisis.

Minor.

No.

seem to grow and then

13

Kohl and Dr.

the

VAN DER EB:

down a little

12

22

there

a short

some cases,

10

16

that

Dr.

--

Yeah. to

know more about

that,

number one. NEAL R. GROSS (202) 2344433

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98

1

DR.

2

DR. KOHL:

3

us about

4

the

the

father

VAN

DER

And, number'two,

neurological

of the

Yeah.

EB:

history

of the

I can,

yes.

6

source

of the serum,

we were able

to

7

--

me see where

I have it

that

8

obtained

in August

of 1985 from

9

serum,

let

and it

was not

10

came from in this

11

samples

12

were all

that

American

15

source.

It

were selected

18

for

,I9

usually

20

either

from Flow or GIBCO or both,

21

one is

the best

And if

22

so that

like

cloning they select

23

enough

24

and we had the other

25

cells

something

for

they

at that

very

time.

GIBCO, also not

European

and they

different

samples

and they test

of human diploid one, the batch

can have enough

serum

of Rotterdam

carefully,

seven

North

These

by the University

of diploid

serum

and afterwards

cells.

samples

Flow

but the Flow serum

sources

17

get

where the

before

these

serum was

was Flow,

was .certainly

we got

back that

the

it

we had sometimes

Yes,

growth

--

case,

American

sources.

16

and

As to the.

trace

stated

we got in the years

Also

14

--

exactly

particular

from North

13

mother

fetus?

DR. VAN DER EB:

5

can you tell

for

half

which cells.

is-large a year,

half.

ACTING CHAIRMAN DAUM:

Thank you.

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99 1

Dr.

2

DR. VAN DER EB:

3

another

please. Oh, prion.

DR. KOHL: the mother

and the

The neurological

7

father.

The mother

8

know and

had

9

mother. the

11

healthy.

--

there

same hospital

The

13

hospital

14

father,

15

abortion

anymore, and that

Both the mother

was completely

in

Leiden,

father

which

was not

was,

in

in

completely

not

to

the

and unknown

reason

why the

was requested.

17

DR. MINOR: it.

Dr. Minor.

You may have said

18

missed

19

number of El in the PER.CG and the site

20

of the DNA? In other

21

to it?

Is

there

anything

words,

22

DR. VAN DER EB:

23

DR. MINOR: in the

Or'is

this

know about

is El really

and I

the

copy

of integration all

there

is

Yeah. it

where it's

actually

-DR. VAN DER EB:

25

afterwards were

the

ACTING CHAIRMAN DAUM:

put

the

down,

fact,

I

with

known,

what was written

That

wrong

two children

16

24

of

and the

normal.

was nothing

She had at least

12

histories

father.

DR. VAN DER EB:

6

10

No, you had

question?

4 5

Minor,

No, El is the only

thing

NEAL R. GROSS (202) 234-4433

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100 1

.. that's

present

that

2

'remember.

3

me.

4

are all

located

5

in that

kind

6

transfection

7

that

8

integrated,

are

in PER.C6.

I can't

Maybe somebody in the audience

I think

about

close

or seven

calcium

I think,

only

nothing

9

that

at this

11

Dr.

12

presentation,

13

adenovirus

transformed

14

transformed

cell

15

their

der

Eb very and

tumor

on.

ia

experimental

19

relate

20

hit

21

-region

22

expressed

23

area

24

explains

25

immunocompetent

tumor

on one chromosome

is

point

Dr.

and it's

another

Cook

to

of

to

per

us

about

tumorigenicity that

and determine

5 to

and

se

like to

to do is focus start

with

models and.how that

of

the

ElA

sensitize

immunological

the lack

informative

tell

at hand and then

ability

interest

to thank

capacity.

tumorigenicity

of Adeno.

going

interactions

development

the

Thank you.

we're

cell

host

to the question about

see after --

DR. COOK: So what I'd

17

may be

only

much for

ask

forming

16

which

So they

phosphate,

one side

it,

else.

I think van

in

you often

ACTING CHAIRMAN DAUM:

10

can correct

copies

to each other.

of tandem repeat with

is,

six

exactly

gene

cells

injury. I

think

of tumorigenicity

talk of

might

a little this

in which That's

it

in

to

El it's

been our

some extent

of these

cells

in

animals. NEAL R. GROSS

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