Long Term Anti-hiv Rnai Therapy

Welcome

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Long Term Anti-HIV RNAi Therapy For AIDS Dr. Rajesh Kumar

Ph.D (Dairy Microbiology) Molecular Biology Unit Dairy Microbiology Division N.D.R.I [email protected]

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INTRODUCTION: HIV/AIDS : major public health problem worldwide.  No effective vaccines are currently available. Although combinatorial therapies such as HAART have proven to be effective, but do not afford a complete cure. Previous approach involved use of transdominant proteins, decoys and ribozymes shown initial promise only.  Intracellular immunization by gene therapy strategies offers a promising approach. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Cont….  RNA interference is a kind of secret weapon inside us.  Silencing genes in HIV is straight forward :- HIT THE VIRUS where it counts. Synthetic siRNAs can specifically and effectively inhibit HIV-I gene expression.  shRNAs :- Powerful tools for long term HIV gene therapy. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

HIV/AIDS Challenges • Continuing spread in Africa, Caribbean • Emerging epidemics • Possible resurgence in high income countries • Anticipated escalation of new infections 40 million individuals infected with HIV in world presently. 28 million infections are preventable. 16000 infections occur worldwide each day. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Global Summary of the HIV/AIDS Epidemic December 2002 Number of people living with HIV/AIDS Total

42 million

Adults

38.6 million

Women

19.2 million

Children under 15 years

3.2 million

Total

5 million

Adults

4.2 million

People newly infected with HIV in 2002

Women

2 million

Children under 15 years

800 000

Total

3.1 million

Adults

2.5 million

AIDS deaths in 2002

Women

3.5 to 4.5 million in India

Children under 15 years

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1.2 million 610 000

Adults and Children Estimated to Be Living with HIV/AIDS as of End 2002 Western Europe North America

570,000

Eastern Europe and Central Asia

1.2 million

980,000 East Asia and Pacific

1.2 million Caribbean

North Africa and Middle East

440,000

550,000

South and SouthSouth-East Asia

6 million

Latin America

1.5 million

SubSub-Saharan Africa

29.4 million Australia and New Zealand

15,000

Total: 42 million pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Molecular and Biological Features of HIV:Member of the lentivirus family of animal retroviruses. HIV variants: a) X4 T cell tropic. b) R5 macrophage tropic. c) dual tropic.

HIV virion consists of two identical strands of RNA (9.2 kb) packaged with in a core of viral proteins.

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Complex Structure of HIV

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HIV-I genome:

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Entry of HIV virion :-

CD4 binding which leads to CCR5 / CXCR4 binding and eventual fusion peptide exposure

CXCR4 or

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HIV Life Cycle Reverse Transcriptase (RT) Inhibitors –AZT (Zidovudine) Entry/fusion inhibitiors –Nevaripine

Protease Inhibitors

Integration

X

Uncoating

CD4 CCR5

Binding & Entry

Reverse Transcription

DS DNA

Genomic RNA

Translation Transcription

viral mRNA

X

X

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Cytoplasm

Assembly & Release

Nucleus

Cytoplasm

Rationale for Gene Therapy for AIDS • The benefits of HAART • The limitations of HAART: Cost—$10,000 to $20,000 per year  Reservoir of latently HIV-infected cells persists despite suppression of replication to undetectable levels  Toxicity  Emergence of drug-resistant HIV strains  Side effects.

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Vaccine against AIDS:  There is currently no vaccine to prevent HIV infection.  Only HIV-negative individuals can volunteer for a preventive HIV vaccine trial.  You cannot become infected with HIV from the vaccines being tested.  All populations must be involved in HIV vaccine research.  Obstacles in the development of a vaccine:-

 Complex structure and life cycle  Genetic potential of the virus for great antigenic variability. International AIDS Vaccine Initiative (IAVI). pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Approaches to Anti -HIV Gene Therapy Anti-HIV The inhibition strategies can be divided into two groups:a) The RNA-based strategies : 

RNA Decoys(sense RNA)

Ribozymes Antisense RNA siRNA RNA aptamers miRNA

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b) Protein-based strategies including : Transdominant negative proteins (TNPs)  Chimeric proteins (fusion proteins)  Nucleases.  Anti-infective cellular proteins.

 Intracellular single-chain antibodies.

 Monoclonal antibodies (Mabs). pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

RNA Decoys

Intracellular Ab & Transdominant Proteins Integration

Uncoating

CD4 CCR5

Binding & Entry

Reverse Transcription

Genomic RNA

DS DNA

Translation Transcription

viral mRNA

Rev

Tat

siRNA Antisense RNA Ribozymes Cytoplasm

Nucleus

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Cytoplasm

Assembly & Release

Double stranded RNA mediated RNA interference  Simple & rapid method of silencing gene expression.  Gene silencing is a consequence of degradation of RNA into short RNAs that activate ribonuclease target homologous mRNA.  It is a two step mechanism:a) First step involves degradation of ds RNA into siRNAs (21-22bp) by Dicer. b) siRNAs join RISC which acts on cognate mRNA & degrades it. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Silencing of Gene Expression by Small Interfering RNA (siRNA)

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Strategies using siRNA to Inhibit HIV-I Replication Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. (Lee NS et al, may 2002.)

 Mammalian Pol III promoter system capable of expressing

functional ds siRNAs following transfection into human cells.  In 293 cells cotransfected with the HIV-1 pNL4-3 proviral DNA and the siRNA-producing constructs, 4 logs of inhibition of expression from the HIV-1 DNA was achieved. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Potent and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference. (Glen A. Coburn & Bryan R. Cullen, sep 2002.)

siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by HIV-1 can specifically block Tat and Rev expression and function. 

 Observations demonstrates thst RNA can effectively block virus replication in human cells.

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Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication. (Martinez MA et al, 2003)

Inhibitory effect directed to CXCR4 was detected in 48 h after transfection of CXCR+U87-CD4+ cells. 

 Expression of CXCR4 & CCR5 was blocked in 63 & 48% of positive cells by corresponding siRNA.  siRNA directed to CXCR4 did not suppress CCR5 expression or vice-versa.

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Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. (Qin XF et al,2003)  Lentivirus based vector designed to introduce siRNAs

against the HIV-I Coreceptor CCR5.  Expression of a potent CCR5-siRNA resulted into upto 10 fold inhibition of CCR5 expression on cell surface.  Lentiviral vector mediated delivery of siRNA proven to be effective. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Targeting CCR5 with siRNAs: using recombinant SV40derived vectors to protect macrophages and microglia from R5-tropic HIV. (Cordelier P et al,2003) Recombinant Tag-deleted SV-40 vectors used to transduce unselected CCR5-bearing cell lines.  SV-40 were designed to express two different anti-CCR5 siRNAs. These siRNAs largely protected CCR5+ cell lines from R5-tropic HIV. Therefore, strategies to target CCR5 using rSV40-delivered, VA promoter-driven siRNAs may be useful therapeutic options for treating HIV infection. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Potent suppression of HIV type 1 infection by a short hairpin anti-CXCR4 siRNA. (Anderson J et al, 2003)

 A stem-loop hair structured anti-CXCR siRNA was designed.  FACS analysis showed marked downregulation of CXCR4.  siRNA transfected cells exhibited marked viarl resistance.  Delivery of these siRNA into hematopoietic stem cells via lentiviral vectors may have potential gene therapeutic applications.

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Bispecific short hairpin siRNA constructs targeted to CD4, CXCR4 and CCR5 confer HIV-1 resistance. (Anderson J et al, 2003)  Previous studies was based on utility of monospecific siRNAs in suppression of HIV-I infection.  High mutation rate considerable challenges in design of fully effective constructs. Bispecific siRNA constructs was designed targeted against CXCR4 or CCR5. Transfected Magi-CXCR4 and CCR5 cells showed significant downregulation of their respective coreceptors.  Results demonstrated the practical utility of short hairpin siRNA bispecific constructs.  it is now possible to introduce promising multivalent siRNA constructs into specific pdfMachinevectors. - is a pdf writer that produces quality PDF files with ease!

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Human Immunodeficiency Virus Type 1 Escapes from RNAi Mediated Inhibition. (Atze T. Das et al, 2004.)  Short-term assays suggested that RNAi may be a powerful new method.  However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays.  In this study siRNA directed against viral Nef gene confers resistance to HIV-1.  siRNA-Nef resistant virus containing substitution or deletion in Nef gene emerged.  Like HAART, antiviral approaches involving RNAi should be used in a combined fashion. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Inhibition of HIV-1 fusion with small interfering RNAs targeting the chemokine coreceptor CXCR4. (Zohu N et al, Dec 2004.)

Immunofluorescence microscopy demonstrated downregulation of CXCR4 with specific siRNAs. Transfections with siRNAs targeting CXCR4 mRNA shown to inhibit HIV-I envelope fusion. The specificity of this effect was demonstrated by the inhibition of fusion by CXCR4-tropic and dual-tropic envelope glycoproteins from HIV-1 on CXCR4+ indicator cells. Targeting cellular cofactor, rather than the HIV-I specific mRNAs holds promise. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector. (Anderson J, Akkina R, Jan 2005)

 Monospecific siRNAs targeting individual coreceptors are inadequate against R5 & X4 viral strains.  Bispecific constructs with dual specificity are required.  Lentiviral vectors incorporating CXCR4 & CCR5 siRNAs of short hairpin design were constructed.  High efficient transduction with a concomitant down regulation was achieved with lentiviral vectors. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Cont…

 siRNA expressing transduced cells demonstrated marked viral resistance against X4 & R5 tropic HIV-I.

 HIV-I resistance was also observed in transduced PBMCs.

 CXCR4 & CCR5 could be simultaneously targeted for downregulation by a single combinatorial lentiviral vector.

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HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. (Ellen M. Westerhout et al, Feb 2005)  HIV-1 replication can be efficiently inhibited by intracellular

expression of an siRNA.  Recently long term inhibitionof HIV-I replication in human T cells stably expressing siRNA-Nef was demonstrated.  However, HIV-1 escape variants emerged after prolonged culturing.  These siRNA resistant viruses contain nucleotide substitution or deletion in or near the target sequence.

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Cont….  Out of 9 escape variants, two virus were found to escape through

mutations that induce an alternative secondary structure of target RNA.  Results demonstrates that occlusion of an siRNA target sequence by RNA secondary structure reduces RNAi efficiency.

 These results highlight the extreme versatility of HIV-I and its evolutionary Capacity to escape from RNAi mediated antiviral therapy.

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Computational Design of Antiviral RNA Interference Strategies That Resist Human Immunodeficiency Virus Escape. (Joshua N. Leonard and David V. Schaffer, Feb 2005.)  Recently developed strategies based upon RNAi appear highly promising and

offer alternative approaches.  RNAi has emerged as a robust and highly evolutionary conserved mechanism for down regulating gene expression.  Despite this the clinical application of RNAi faces a number of challenges: Certain type of siRNA can induce considerable off-target changes in gene expression.  Exquisite sequence specificity of RNAi makes the problem of viral escape.  Optimization is required in the design of effective antiviral RNAi therapies.  Computational model of HIV-I replication used to elucidate design principles for applying RNAi against HIV-I in a manner that delays viralpdfMachine escape.- is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Contd….. A novel type of computer model:  MASS simulation that incorporates molecular level details of HIV-I reproduction and susceptibility to RNAi.  This system could be used to study more fundamental aspects of viral evolution.  Increasing no of RNAi targets even while keeping siRNA level constant dramatically mproves RNAi efficiency.  This approach can yield insights into highly complex process by which replicating entities evolve.

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Lentiviral siRNAs targeting multiple highly conserved RNA sequences of HIV-I.

(Chang LJ, Liu X, HeJ, Jul 2005)

 High mutation rate of HIV makes it difficult for any therapy to

sustain prolonged effect.  To explore novel therapy, lentiviral siRNA vectors targeting multiple highly conserved regions in HIV-I genome was devloped & tested.  siRNA expression cassette was cloned into self-inactivating insulator vector.  Some siRNA targeting sites were also present in the helper construct of vector system.  siRNA targeting gag, pol and vpu but not nef efficiently inhibited replication of pNL4-3(subtype B), p89.6 (subtype B) &pdfMachine p90CF402.1.8 - is a pdf writer that(subtypeA/E). produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Cont….  Transduction of a long term chronically infected human lymphoma cell line with lentiviral siRNAs resulted in stable inhibition of HIV-I replication.  Northeren analysis showed that both genomic & subgenomic viral RNA species were downregulated.  Inhibition of viral RNA persisted after prolonged passage.  Using these vectors reduced replication kinetics of HIV-I in human PBLs was demonstrated.  Lentiviral siRNAs targeting multiple conserved HIV-I sequences holds significant promise for the treatment of HIV-I infections. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

CXCR4 and CCR5 shRNA transgenic CD34+ cell derived macrophages are functionally normal and resist HIV-1 infection. (Joseph (Joseph Anderson Anderson and and Ramesh Ramesh Akkina, Akkina, Aug Aug 2005) 2005)

 RNAi can effectively down regulate the expression of either viral or cellular RNA.  The potency of shRNAs in targeted gene silencing qualifies them as powerful tools for long term HIV gene therapy.  A no of reports have shown, delivery of siRNAs by transfection of presynthesized siRNAs into cultured cells can effectively inhibit HIV-I replication.  Due to transient nature of transfected nucleic acids, the antiviral effects are only temporary.  For long range therapy prolonged maintenance & expression of siRNA coding transgenes in a susceptible target cell is necessary. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Cont….

 In this regard lentiviral vectors have proven to highly effective in high efficiency gene transduction & sustained gene expression.  Targeting HIV-I genes alone will not be sufficient due to high possibility of generating escape mutants.  More sustained efficacy of antiviral effects may be obtained by targeting host cellular genes critical for viral entry and/or replication.  CXCR4 and CCR5 play critical roles as coreceptors for viral entry. CXCR4  T cell tropic X4 HIV-I. CCR5  Macrophage tropic R5 HIV-I. Their sustained knock down may prove to be more efficacious for long range siRNA therapy. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Cont…

 Considering both coreceptors will be imp in developing effective therapeutics.  Individuals carrying naturally occurring 32-bp deletion in the CCR5 gene results function thus conferring significant resistance to HIV infection.  Homozygous or heterozygous individuals with this mutation remain physiologically normal.  Based on this rationale, recent work with synthetic siRNAs demonstrated that down regulating either CXCR4 or CCR5 will protect cells from X4 or R5 HIV-I strains, respectively, at the level of viral entry.  Down regulating CCR5 alone in the face of an HIV-1 infection is insufficient. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Cont…

 Therefore synthetic bispecific combinatorial constructs as well as a bispecific lentiviral vector targeting both CXCR4 and CCR5 showed efficacy in inhibiting HIV-1 infections in cell culture lines.  In translating these findings into a stem cell gene therapy setting, this bispecific lentiviral vector was used to generate shRNA expressing transgenic macrophages.  Macrophages, along with T cells, are major cell targets of HIV infections.  Programming these cells to express shRNAs targeted CXCR4 and CCR5, could confer resistance to HIV infection.  shRNAs can have possible off target effects thus transgenic macrophages also need to be assessed for proper functionality. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

CXCR4 and CCR5 bispecific siRNA lentiviral vector XHR  Transfer vector pHIV-7-GFP was designed to contain an antiCXCR4 shRNA cassette under the control of the Pol-III U6 promoter and an anti-CCR5 shRNA cassette under the control of the Pol-III H1 promoter. anti-CXCR4------targets--- CXCR4 transcript at 3-23 nt. anti-CCR5--------targets-- CCR5 transcript at 13-31 nt.  Two cis-acting elements used to enhance the performance of vector, namely:a) central DNA flap. b) WPRE.  This vector also contains an EGFP reporter gene downstream from shRNA cassettes.

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Bispecific Bispecific lentiviral lentiviral vector vector (XHR) (XHR) encoding encoding anti-CXCR4 anti-CXCR4 and and CCR5 CCR5 shRNAs. shRNAs.

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Results:(I) Lentiviral vector transduction of CD34+ cells with CXCR4 and CCR5 shRNAs and derivation of mature macrophages.  CD34+ hematopoietic progenitor cells transduced with either control GFP or XHR vectors sorted for EGFP and driven towards a myeloid lineage in semi-solid methyl cellulose cytokine media to generate transgenic macrophage.  No significant differences were found in the levels of macrophages obtained when compared between the control GFP vector and XHR vector transduced cells or control non-transduced CD34+ cells.  The morphology of the transgenic macrophages also appeared normal (data not shown). pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

(II) Down regulation of HIV-1 coreceptors

CXCR4 and CCR5 in transgenic macrophages.  In XHR transduced cells FACS analysis showed an 82%

decrease in CXCR4 expression.  GFP-alone control vector transduced cells and nontransduced cells displayed normal levels of CXCR4 expression (94%).  Similar analysis for CCR5 expression (75% decrease in transduced macrophages)  Thus, stably transduced macrophages exhibited significant down regulation of both the coreceptors CXCR4 and CCR5 due to shRNA targeting. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

FACS Analysis

Fig II.  Down regulation of the coreceptors CXCR4 and CCR5 in XHR transgenic macrophages. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

(III) XHR transgenic macrophages resist HIV-I

challenge.  Transduced macrophages were challenged with X4-

tropic (NL4-3) and R5-tropic (BaL-1) strains of HIV-1.  Antigen ELISA to detect viral p24 in culture supernatants were performed on various days postinfection.  Over a 2-log reduction in viral yield was seen in XHR transduced macrophages compared to control cells.  Thus stable coreceptor down regulation by siRNAs resulted in marked protection of transgenic macrophages against viral challenge.

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1-log reduction in viral yield. 2-log reduction in viral yield.

Fig III.  HIV-1 resistance of XHR transgenic macrophages

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(IV) Transgenic macrophages display characteristic phenotypic cell surface markers.  Off target effects of some siRNAs may disrupt the phenotypic properties of macrophages.  Therefore, transgenic macrophages were subjected to phenotypic analyses to assess their characteristic cell surface markers by FACS.  Levels of the monocyte/macrophage marker CD14 in XHR macrophages were found to be similar to GFP-alone transduced or nontransduced cells (98% and 97% respectively).  Similarly the levels of CD4 were found at comparable levels for XHR and GFP-alone transduced macrophages at 95% and 93% respectively.

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Fig IV.  Transgenic macrophages display normal cell surface markers. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

(V) Transgenic macrophages are functionally normal.  Some shRNAs could have possible off-target global effects leading to disruption of normal cellular functions.  Functional assays on transgenic macrophages to evaluate this possibilty were performed.  Macrophages are typically antigen presenting cells which are phagocytic in nature as well.  To determine if XHR transgenic macrophages retained the phagocytic function, they were presented with fluorescently labeled E. coli (Bioparticles).  Foreign cell uptake was measured by FACS.  No significant difference in phagocytic capacity were found. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Contd…

 Based on fluorecscence levels, XHR macrophage phagocytosis was quantified at 68.2% compared to non transduced and GFP-alone cells at 63.5% and 61.5%, respectively.  Transduced Magi-CXCR4 cells, serving as non-phagocytic cell controls did not display any phagocytic activity.  Macrophages secrete and respond to a number of important cytokines that include IL-1 and TNF-á.  To determine functional capacity, siRNA transgenic macrophages were stimulated with LPS.  No significant differences were seen in levels of IL-1 and TNF-á cytokine secretion among the transgenic and control cell types.  These finding showed that siRNA transgenic macrophages are phenotypically and functionally normal. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

0%

63.5%

61.5%

68.2%

Fig V (a)  Phagocytosis of fluorescently labeled E.coli by CD34+ derived macrophages pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

LPS stimulation

Fig V (b)  XHR transgenic macrophages secrete normal levels of the cytokines IL-1 and TNFá. pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

CONCLUSION 

siRNAs are recognized as powerful reagents to reduce the expression of specific genes.



To use siRNAs as reagents to protect cells against viral infection, effective methods for introducing siRNAs into primary cells are required.



Stable simultaneous knock down of both the coreceptors CCR5 and CXCR4 is necessary to prevent HIV-1 infection at the entry level by both R5 and X4, as well as dual tropic viral strains.



Present studies have demonstrated that a bispecific lentiviral vector could be used to stably deliver shRNAs targeted to both CCR5 and CXCR4 coreceptors into CD34+ hematopoietic progenitor cells and derive transgenic pdfMachine - is a pdf writer that produces quality PDF files with ease! macrophages. Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

Contd…..



Stable down regulation of both the coreceptors was achieved in transgenic macrophages.



The siRNA expressing macrophages were also found to be phenotypically and functionally normal.



It is now possible to construct gene therapeutic lentiviral vectors incorporating multiple siRNAs targeted to cellular molecules that aid in HIV-I infection.



Use of these vectors in a stem cell setting shows great promise for sustained HIV/AIDS gene therapy.

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CD4+ T cell

CD4+CD8+

CD8+ T cell

lymphoid stem cell pre-B cell

B cell

plasma cell

dendritic cell

Pluripotent Stem Cell CFU-M multilineage myeloid stem cell (CFU-S)

monocyte

CFU-G/M CFU-G PMN

E/mega

erythrocyte

BFU-E CFU-E

CFU-Mega pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

megakaryocyte

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