Cell Biol Virus Vectors Aug 2009

Gene Transfer by Animal Viruses & Introduction of Gene into Cell 2552

วาสนา ศิรริ ังษี 25 สิงหาคม 1

Objectives: after finish this class, student should be able to explain these topics;  

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How virus acts as a genetic vector. Various procedures in performing gene transfer (non-viral & viral vector). Types of viral vectors. Viral vector construction. Advantages and disadvantages of specific viral vector. Viral vector application in medicine. 2

Viruses as vectors 

Viruses are obligate intra-cellular parasites which infect cells, often with great specificity to a particular cell type. They tend to be very efficient at transfecting their own DNA into the host cell, which is expressed to produced new viral particles. By replacing their non-essential genes with foreign genes of interest, the recombinant viral vectors can transduce the cell type it would normally infect. 3

Gene transfer into eukaryotic Transduction: is the process by which DNA is transferred cells to cell or bacterium by a virus/bacteriophage using natural

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process of viral infection. Common techniques are the use of viral vectors.

Transfection: is a process to introduce naked DNA into cell culture by various procedures. The mostly used procedure is that cells are treated with calcium phosphate or DEAE-dextran that increase cell permeability. Other procedures are --lipofection (DNA is bound to positively charged lipids; cationic liposome) that are capable of fusing with the lipid bilayer of the cell membrane. --electroporation 4

Electroporation Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field.

Cuvette

ลักษณะของผนั งเซลล์: ปกติ ปล่อยกระแสไฟฟ้ า (รูพรุน) หยุดกระแสไฟฟ้ า กลับมาปกติ

หลัง 5

Gene transfer (con.) 

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Microinjection: microinject directly DNA into the cell’s nucleus. The nuclei of oocytes and eggs are well suited for this approach. High energy bombardment: DNA is coated on the gold particle and is shot directly into the cell.

Molecular conjugates: DNA is bound to protein or synthetic ligands. Targeting proteins include asialoglycoprotein, transferin, and polymeric IgA. Delivery technique is similar to those for liposomes. 6

Types of viral vectors 

RNA virus vectors Retrovirus, Lentivirus

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DNA virus vectors Adeno-associated virus

(AAV; Human Parvovirus),

Adenovirus, Herpes simplex virus, Poxvirus (vaccinia virus, canarypox virus)

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Comparison of genetic vectors Vector

Insert size (kb) Persistence

Non-viral

Unlimited

Advantages

Disadvantages

Transient

No viral sequence, large capacity

Low-efficiency entry, transient persistence

Target cell division required

Retrovirus

1-7.5

Stable

Effective cell entry, stable integration

Lentivirus

1-8

Stable

Transduce deviding & Biosafety profile nondividing cell

AAV

4 (±5%) Stable Replication defective

Adenovirus

2-35

HSV Poxvirus

Unknown (genome 150kb) 25

recombinant production

Efficient cell entry packaging size

uncertain

Limitation on

Transient

Effective in vivo gene delivery

Transient persistence

Stable in neuron

Neuronotropic large capacity

Complex virus

Stable

Cheap, heat-stable,

Low efficiency stimulate HMI&CMI vector 8

RNA Viruses: (1) Retroviruses are a class of enveloped viruses containing Retroviral vector a duplicated ssRNA genome. 

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Following infection, the viral genome is reverse transcribed into ds DNA, which integrates into the host genome and is expressed as proteins. The viral genome is approximately 10kb, containing at least three genes: gag (coding for core proteins), pol (coding for enzymes; reverse transcriptase, protease, integrase) & env (coding for the viral envelope protein). At each end of the genome are long terminal repeats (LTRs) which include promoter/enhancer regions & sequences involved with integration and sequences required for packaging the viral DNA. 9

Retroviral vectors are most frequently based upon the Moloney murine leukaemia virus (Mo-MLV), which is an amphotrophic virus (infect mouse & human cells), enabling vector development in mouse models, & human cells, enabling human treatment. A requirement for retroviral integration and expression of viral gene is that the target cells should be dividing. This limit gene therapy to proliferating cells in vivo or ex vivo, whereby cells are removed from the body, treated to stimulate replication and then transduced with the retroviral vector, before returning to the patient.

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Ψ+ A

gag

5’ LTR

pol

env

3’ LTR

Ψ+ B

5’ LTR

Exogenous gene (s)

3’ LTR

Map of typical simple retrovirus and retroviral vectors. A: the retrovirus has an LTR at each end, Ψ: Psi, the encapsidation sequence, and region encoding the Gag, Pol, and Env polyproteins. B: retroviral vectors containing the LTR and Ψ+ regions of the retrovirus with the exogenous gene sequences cloned in between.

Vectors lacking the genes encoding the retroviral proteins are replication defective and shouldn’t be capable of repeated replication after transduction of target cells. 11

Production of retroviral vector

1

2

No packaging signal

(Virus contains gene of interest)

Mouse embryo fibroblast cells

1. Production of packaging cell: cell that expresses viral proteins but lacks packaging signal. 2. Transfection of “LTR + gene of interest + packaging signal” to packaging cell. 12

Retroviral vector life cycle Recombinant vector plasmid (dsDNA) Transfect into packaging (producer cell) Production of viral vector

Integrated or episomal DNA (dsDNA) in packaging cell Transcription Recombinant viral genome (ssRNA) Translation

Package into virion & release virus vectors from producer cell Enter target cell Production of gene product

Reverse transcribe

Integrate

Provirus (dsDNA) in target cell Transcription Transcripts (ssRNA) Translation Gene product (Protein of interest)

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Limitation of retroviral vector: Some retroviruses contain proto-oncogenes, which when mutated can cause cancers, however, in the production of vectors these are removed. Retroviruses can also transform cells by integrating near to a cellular proto-oncogene and driving inappropriate expression from the LTR, or by disrupting a tumour suppresser gene. This event, termed insertional mutagenesis, though extremely rare could still occur when retroviruses are used as vectors.

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(2) Lentivirus vectors 

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Lentiviruses are a subclass of retroviruses which are able to infect both proliferating & non-proliferating cells. They are more complicated than simple retroviruses, containing basic gag-pol-env genes and additional 6 proteins, tat, rev, vpr, vpu, nef & vif. The lentiviral vectors used are derived from the HIV & are being evaluated for safety, with a view to removing some of the non-essential regulatory genes.

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Applications 

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Because of the long and stable insertion of gene into the host genome, retroviral vectors were the earliest vectors applied to gene therapy. The first human gene therapy protocol involved removal of peripheral blood cells from 2 children with adenosine deaminase (ADA) deficiency, in vitro transduction with a retroviral vector encoding ADA, and reintroduction of the cells into children. A further development of ADA gene therapy was transduction of hematopoietic stem cells with an ADA-expressing retroviral vector, resulting in expression in multiple hematopoietic lineages and clinical improvement in the patients. Because of ability to stably transduce nondividing cells, lentiviral vectors have proved particular interesting for application in the nervous system (e.g., Parkinson’s disease). 16

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DNA viruses: (1) AdenoAssociated Viruses (AAV) 

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Adeno-associated viruses (AAV) are defective nonpathogenic human parvoviruses, dependant on a helper virus, usually adenovirus, to proliferate. They are capable of infecting both dividing & non dividing cells. They can integrate into a specific point of the host genome (human chromosome 19) at a high frequency. The wild type genome is a ssDNA molecule, consisting of 2 genes; rep, coding for proteins which control viral replication, structural gene expression & integration into the host genome, and cap, which codes for capsid structural proteins. 18

AAV genome

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At either end of the genome is a 145 bp terminal repeat (TR), containing a promoter. When used as a vector, the rep & cap genes are replaced by the transgene and its associated regulatory sequences. The total length of the insert cannot greatly exceed 4.7 kb, the length of the wild type genome. Production of the recombinant vector requires that rep & cap are provided in trans, along with helper virus gene products (E1a, E1b, E2a, E4 & VA RNA from the adenovirus genome). 19

AAV life cycle

Absence of helper Adenovirus

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Advantage: - AAV vectors integration into the host genome allowing prolonged transgene expression. - Gene transfer into vascular epithelial cells, striated muscle & hepatic cells, with prolonged expression when the transgene is not derived from a different species. - Neutralizing antibody to the AAV capsid may be detectable, but does not prevent re-administration of the vector or shut down promoter activity.

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Gene Therapy for cystic fibrosis

(AAV)

Produce normal mucus

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(2) Adenovirus 

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Adenoviruses are non-enveloped viruses containing a linear ds DNA genome. While there are over 40 serotypes of adenovirus, most of which cause human respiratory tract infections. Subgroup C serotypes 2 or 5 are predominantly used as vectors. The life cycle does not normally involve integration into the host genome. Replicate as episomal elements in the nucleus of the host cell & consequently there is no risk of insertional mutagenesis. The wild type adenovirus genome is approx. 35 kb of which up to 30 kb can be replaced with foreign DNA.

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Adenovirus vector construction Adenovirus genome contains early genes (E1a, E1b, E2, E3 & E4), which have regulatory functions, and a late genes L1-L5, which codes for structural proteins.

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 Adenoviral vestors are very efficient at transducing target cells in vitro & vivo, & can be produced at high titer (>1011 /ml).  The initial delivery of large amounts of DNA packaged within adenovirus proteins, the majority of which will be degraded & presented to the immune system may still cause problems for clinical trials.  The development of vectors containing fewer genes, in the "gutless" vectors which contain no viral coding sequences, has resulted in prolonged in vivo transgene expression in liver tissue. 26

Until recently, the mechanism by which the adenovirus targeted the host cell was poorly understood. Tissue specific expression was only possible by using specific cellular promoter/enhancers. e.g. - the myosin light chain 1 promoter - the smooth muscle cell SM22a promoter or by direct delivery to a local area.

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Applications 

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To date, >170 clinical trials with Ad vectors have been performed in humans. Monogenic diseases (an inherited disease controlled by a single pair of gene) – Ex. Cystic fibrosis

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Cancer- Ex. Delivery of a wide range of cancer therapeutic genes, including a) genes that encode immunostimulatory molecule such as IL-2; b) tumor suppressor genes such as p53; c) suicide genes such as HSV-Thymidine Kinase gene.

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Vaccine – vaccine delivery vector against HIV, Ebola virus, and Plasmodium.

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(3) Herpes simplex virus 

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Herpes simplex virus type 1 (HSV-1) is a human neurotropic virus. HSV-1 as a vector for gene transfer to the nervous system. The wild type HSV-1 virus is able to infect neurons & either proceed into a lytic life cycle or persist as an intranuclear episome in a latent state.

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The viral genome is a linear ds DNA molecule of 152 kb. There are two unique regions, long & short (termed UL & US) which are linked by internal repeat sequences (IRL & IRS). At the non-linker end of the unique regions are terminal repeats (TRL & TRS). TRL

UL

IRL

IRS

US

TRS

There are up to 84 genes of which about half are not essential for growth in cell culture. Once these non essential genes have been deleted, 40-50 kb of foreign DNA can be accommodated within the virus. 30

Structure of HSV-1 genome and viral vector capacities

Comparison between different viral vector capacities

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HSV-1 as efficient gene  HSV-1 has a large ds DNA genome containing many transfer vector non-essential genes which can be deleted without 

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reduction of virus yield, providing space for insertion of either large single gene or multiple transgenes. Virus possesses a natural mechanism for maintenance of the viral genome as an episome in nucleus of latently infected nerve cells. Virus possesses a novel promotor system that is active during latency. Virus has a broad host range. HIV-1 is able to transduce both dividing and nondividing cells. Even highly deficient virus recombinant can be propagated to the high titers (>1010 PFU/mL) required for in vivo gene therapy application. 32

Applications 

Neurobiology- gene transfer to the peripheral nervous system for treatment of drug-induced peripheral neuropathies.

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Oncology- Oncolytic HSV-1 vectors have been tested for tumor-specific delivery of antineoplastic gene products, including immunoregulatory molecule, pro-drug converting enzymes, and angiogenesis inhibitors.

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Vaccine – Gene to be expressed from an HSV vector was the HBV surface antigen, with the idea of using the recombinant virus as a vaccine for HBV.

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(4) Poxviruses 

In 1982, it was suggested that since vaccinia virus had been widely used and proven safe (against smallpox), it could be used as a vector for carrying foreign antigens and thereby immunizing against other pathogens.

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To date, hundreds of different vaccinia virus recombinants have been described.

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Advantages of recombinant vaccinia virus vector vaccines: • cheap to manufacture and administer, • stable - do not require refrigeration, stimulate both HMI & CMI

Disadvantages: • Vaccinia can lead to death in immune deficient individuals. • To overcome this disadvantage, avian poxviruses (fowlpox and canarypox) are now being used as vectors. • These viruses can infect mammalian cells but cannot produce new infectious virus in mammalian hosts.

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Basic strategy for Vaccinia  virus vector 1. Plasmid construction: Create a gene construct under the vaccinia promoter control. The foreign hybrid gene can be inserted in a non-essential site in the vaccinia genome: e.g. the thymidine kinase (TK) gene is the most popular site of insertion because the virus does not need TK for replication. 

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2. Transfect a gene construct and a wild-type vaccinia virus into a eukaryotic tissue culture cell. 3. Viral vector selection: TK (-) virus is less pathogenic and bromodeoxyuridine (BUDR) resistant. TK (+) wild type virus is BUDR sensitive. 36

Vaccinia virus vector Foreign gene construction Plasmid construction

Transfection into cells

Production of Vaccinia virus carrying foreign gene which is BUDR Resistant (TK-). 37

Application 

Live vector vaccine - HIV-1 vaccine (Prime-Boost protocol) (Phase III trial in Thailand: start Oct 2003, 16,000 volunteers)

“ALVAC, canarypox vector vaccine” (ALVAC at weeks 0, 4, 12, 24 boost by AIDSVAX B/E a gp120 protein vaccine at weeks 12, 24 follow-up 3 yrs after vaccination)

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Assignment: Write 1 page essay of up to date the most advanced; -virus vector vaccine or -virus vector for gene replacement therapy. References should be included. Deadline: September 16, 2009.

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