Targets & Tools for anti-hiv Gene Therapy. Paula Cannon, PhD University of Southern California

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1 Targets & Tools for anti-hiv Gene Therapy Paula Cannon, PhD University of Southern California

2 Why is HIV a good candidate for gene therapy It s an incurable, serious, life-long infection It s expensive to treat: <$316,000 lifetime cost of drug, 2005 estimate (Shackman 2006) <84% AIDS healthcare costs are drugs (Chen 2006) It infects hematopoietic cells relatively accessible cell types It integrates into our genomes, so like a genetic disease Actively engaged, well-informed patients, prepared to enroll in clinical trials

3 Why can t drugs cure HIV?

4 HAART suppresses HIV to undetectable levels HAART Rapid decay of virus Limit of detection Virus should eventually die out?

5 HAART suppresses HIV to undetectable levels HAART Rapid decay of virus In reality, low levels of virus persist in long-lived reservoirs Limit of detection

6 Cessation of HAART leads to rapid viral rebound Although HAART we can come close to arresting disease in many people, we can not eradicate the virus, and drug therapy needs to be extended indefinitely Viral rebound Further complications Chronic adherence is difficult Drug resistance develops Long-term effects of both drugs & virus on health Politics and economics

7 Why is HIV not a good candidate for gene therapy If a long-lived and latent reservoir is preventing drugs from eradicating HIV, likely also to be a problem for stem cell or gene therapies HIV rapidly mutates and may evolve resistance to therapies Genetic and cellular therapies are complicated, first-world medicines that won t work. You only need to see one talking dog to believe that dogs can talk. [Rick Loftus]

8 The Berlin Patient - the remarkable case of an HIV+ man who was cured by a stem cell transplantation Timothy Ray Brown

9 Viral targets Viral RNA Viral DNA Integrated Provirus

10 Viral targets PR -proteolytic maturation Env - entry Viral RNA RT- reverse transcription Viral DNA IN - integration Integrated Provirus Tat - transcription Rev -RNA export

11 Viral targets Env - entry Fuzeon PR -proteolytic maturation Ritonavir AZT Viral RNA RT- reverse transcription Viral DNA Raltegravir IN - integration Integrated Provirus Tat - transcription Rev -RNA export

12 Viral targets Env - entry Fuzeon PR -proteolytic maturation Ritonavir AZT Viral RNA RT- reverse transcription Viral DNA Raltegravir IN - integration Integrated Provirus Tat - transcription Rev -RNA export

13 Tat and Rev early targets of gene therapy - Tat binds to TAR, an RNA structure at start of all HIV transcripts, and promotes transcription elongation - Rev binds to RRE, an RNA structure in all unspliced HIV mrnas, and promotes export of those RNAs to the cytoplasm Tat TAR RNA pol II Viral RNA transcripts Gene Therapy TAR Strategies: and RRE decoys Ribozymes against Tat shrnas against Tat/Rev Rev TD protein (RevM10) TAR decoy

14 Cellular targets Maraviroc CCR5 co-receptor Viral RNA Viral DNA Integrated Provirus

15 The CCR5Δ32 mutation and HIV resistance CCR5 at cell surface CCR5Δ32 is truncated and trapped intracellularly - CCR5Δ32 homozygotes almost completely resistant to HIV-1 - Phenotype otherwise insignificant

16 Cellular targets Host cell anti-viral restriction factors Viral RNA Viral DNA Integrated Provirus

17 Cellular restriction factors LTR Gag Vif Rev Tat LTR Pol Vpr Vpu Env Nef Restriction Factor Target Accessory HIV accessory Protein protein Gene Therapy Strategy: Express HIV-resistant versions of restriction factors eg rhesus APOBEC3G cannot be degraded by HIV-1 Vif Vif degrades APOBEC3G (SCF Ub ligase) Vpu degrades CD4 (SCF Ub ligase), removes tetherin from cell surface Nef reroutes MHCI (AP-1) and CD4 (AP-2) to lysosomes Vpr degrades UNG2 (Cul4-DDB1 Ub ligase)

18 What cells should we be engineering?

19 Target cell choice Easy to harvest and engineer T cells Hematopoietic stem cells (HSC) Macrophages

20 Target cell choice Easy to harvest and engineer T cells Hematopoietic stem cells (HSC) Longer lasting, impact more cell types Macrophages

21 Animal models to evaluate anti-hiv therapies

22 Animal models Making HIV-permissive small animals has been difficult Mice, rabbits, cats Other lentiviral infections Cats infected with FIV Simian models Chimps can be infected with HIV African or Asian monkeys can be infected with SIV or SHIV Humanized mice

23 Humanized mice Immunodeficient mouse strains NOG: NOD/SCID/IL-2Ry NSG: NOD/SCID/IL-2Ry null RAG: BALB/cA-rag2 null NRG: NOD/SCID/rag1 null/ IL-2Ry null IL-2, IL-4, IL-7, IL-9, IL-15, IL-21 γc X-SCID

24 Humanized mice human CD34+ HSC Human cells throughout mouse tissues SSC CD45 65 bone marrow 83 NOD/SCID/IL-2Rγ null (NSG) mouse CD8 CD4 thymus spleen CD8 CD4

25 Humanized mice human CD34+ HSC NOD/SCID/IL-2Rγ null (NSG) mouse HIV 1 a sm la p l s/m ie p c o A N R week post infection CXCR4 virus CCR5 viruses

26 Summary of HIV gene therapy trials to date

27 T cell trials Phase Vector Anti HIV gene T cells (autologous) 1 2, 2 retrovirus CD4 CD3 signaling chain ζ CD4 and CD8 1 2 retrovirus RevM10 CD4 1 retrovirus Tat ribozyme CD4 (syngeneic) 1 retrovirus RevM10 and TAR antisense CD4 (syngeneic) 1 retrovirus U5 ribozyme CD4 1 2, 2 lentivirus Env antisense CD4 1 retrovirus gp41 peptide fusion inhibitor CD4 1 (ongoing) adenovirus ZFNs against CCR5 CD4 Adapted from Rossi, June & Kohn, 2007

28 HSC trials Phase Vector Anti HIV gene HSC source (autologous) 1 retroviral RevM10 Mob. blood 1 retroviral RRE decoy Bone marrow 1 retroviral RevM10 Bone marrow 1 retroviral Tat ribozyme Mob. blood 2 retroviral Tat ribozyme Mob. blood 1 (ongoing) lentiviral Tat/Rev shrna, TAR decoy, CCR5 ribozyme Mob. Blood Adapted from Rossi, June & Kohn, 2007

29 Experience to date Gene therapy trials have demonstrated feasibility and safety of engineering T cells and HSC However, efficacy has been limited - Low levels of modified cells - Limited potency of therapeutic genes - Requirement for long-term expression of anti-hiv transgenes Some suggestion of preferential survival of modified T cells and progeny of HSC

30 The Berlin Patient Wuff! Timothy Ray Brown

31 HSC transplantation for leukemia HSC donor chemotherapy HSC Leukemia or lymphoma patient

32 HIV+ CCR5Δ32 donor HIVchemotherapy

33 HIV RNA HAART

34 The caveats He was fully ablated high dose chemotherapy plus radiation Also given potent immunosuppression to prevent graft versus host disease including antithymocyte globulin (ATG) that is a potent mix of anti-t cell antibodies - Did this result in depletion of the long-lived viral reservoir? - Was there a donor immune response against the patient s HIV-infected cells?

35 HIV+ CCR5Δ32 donor HIVchemotherapy

36 Ex vivo HSC engineering HIV+ HIV-? Patient s own cells made CCR5-

37 Functional cure HAART No (or limited) viral rebound

38 Gene therapy tools RNA - shrna, ribozyme, antisense, aptamers - Decoys of RNA structures TAR, RRE Proteins - TD viral proteins eg RevM10 - Modified resistance factors - Fusion inhibitors Suicide genes (Tat inducible) Gene knockout or modification -AAV - Targeted nucleases eg ZFNs, TALNs

39 Zinc finger nucleases COOH Zinc fingers bind DNA residues at tip of finger can be modified for a custom fit NH2 Adding nuclease domain creates ZFN that will cut DNA Linking fingers lengthens DNA binding site (4 fingers = 12bp) GCGATTAACGAG ATGCATGGATCCGAGA CGCTAATTGCT CTACGTACCTA GGCTCT Nuclease domains dimerize, further extending DNA target site

40 How zinc finger nucleases disrupt the CCR5 gene CCR5 gene DNA break NHEJ error prone repair CCR5Δ32 truncation Small insertions, deletions disrupt CCR5 ORF ZFN target

41 Humanized mice human CD34+ HSC NOD/SCID/IL-2Rγ null (NSG) mouse HIV 1 a sm la p l s/m ie p c o A N R week post infection CXCR4 virus CCR5 viruses

42 Evaluating CCR5 knockout in HSC by ZFNs human CD34+ HSC NOD/SCID/IL-2Rγ null (NSG) mouse HIV 1 a sm la p l s/m ie p c o A N R? week post infection ZFN-treated HSC

43 ZFN-treated HSC generate HIV-resistant cells in vivo HIV-1 infected Ctrl. Ctrl. ZFN bone marrow thymus CD8 SSC CD45 R5 Ctrl. ZFN CD4 spleen CD8 CD4 intestine SSC CD3

44 Clinical evaluation of CCR5 ZFN modified cells SB-728 Adenovirus CCR5 ZFNs T cells apheresis enrich CD4+ expand cryopreserve test T cells, Phase I infuse ENDPOINTS safety CD4/HIV levels CCR5 selection

45 Clinical evaluation of CCR5 ZFN modified cells SB-728 Adenovirus CCR5 ZFNs T cells apheresis enrich CD4+ HSC G-CSF mobilize, apheresis, CD34+ purify SB-728 Adenovirus CCR5 ZFNs expand cryopreserve test T cells, Phase I chemotherapy HSC, AIDS lymphoma (preclinical stage) cryopreserve test infuse ENDPOINTS safety CD4/HIV levels CCR5 selection infuse

46 Summary Gene targets for HIV both viral and host factors Cellular targets both T cells and HSC Trials to date have shown no/modest effects Reasons to be optimistic - better vectors/protocols - more potent anti-hiv genes - combinatorial therapies - selection for modified HSC - gene knockout technologies applicable to CCR5

47 Acknowledgments USC Ursula Hofer Colin Exline Jill Henley Charles Zampila Kathy Burke Nat Holt Sangamo Biosciences Jianbin Wang Mike Holmes Philip Gregory City of Hope Dave DiGiusto John Zaia John Rossi 5P01 HL Disease team DR1-1490