Curing HIV: Coping with Hope. Ronald Mitsuyasu, MD Professor of Medicine UCLA Center for Clinical AIDS Research and Education (CARE Center)

Transcription

1 Curing HIV: Coping with Hope Ronald Mitsuyasu, MD Professor of Medicine UCLA Center for Clinical AIDS Research and Education (CARE Center)

2 Curing HIV is: 0% A.Impossible 0% 0% 0% 0% 0% B. Requires removing all HIV from the host C. Means controlling HIV without antiretroviral therapy D.Requires high dose chemotherapy E. Is only possible in newborns F. B + D

3 Possible approaches to curing HIV are: 0% 0% 0% 0% 0% A. Continuing ART for > 70 yrs with no missed doses B. Requires inducing HIV from latent reservoirs C. Only occurs with bone marrow or stem cell transplants D. Will likely require multiple strategies E. Means the person cannot be infected again with HIV

4 Antiretroviral Drugs 2014 (28) Reverse Transcriptase Inhibitors(13) Protease Inhibitors (10) Nucleosidetide analogues zidovudine (zdv) didanosine (ddi) zalcitabine (ddc) stavudine (d4t) lamivudine (3TC) abacavir (ABC) emtricitabine (FTC) tenofovir (TFV) Non-nucleoside analogues nevirapine (NVP) delavirdine (DLV) efavirenz (EFV) etravirine (ETV) rilpivirine (RPV) Integrase Inhibitor (3) raltegravir (RAL) elvitegravir (ELV) dolutegravir (DTG) Fusion Inhibitor Infuvirtide (Fuzeon) Entry Inhibitor (CCR5) maraviroc (MVC) saquinavir (SQV) ritonavir (RTV) indinavir (IDV) nelfinavir (NFV) amprenavir (APV) lopinavir/r (LPV/r) fosamprenavir (FPV) atazanavir (ATV) tipranavir (TPV) darunavir (DRV)

5 Problems with Current ARV Therapies Must be taken continuously Does not eradicate HIV. Only inhibits replication There are side effects and toxicities with each class of ARV drugs Difficulty with long term adherence (fatigue factor) Resistance development Expense and inconvenience of chronic therapy

6 Major Problem with ART Federal Resources / Policies/Issues : HIV/AIDS Care Continuum

7 What Do We Mean By Cure? Sterilizing Cure Eradication of all HIV from the individual No HIV found in any tissue (blood, BM, LN, GI, CSF) Cannot detect HIV by most sensitive assays No recurrence of HIV upon treatment interruption Functional Cure No measurable HIV RNA in blood after long term treatment interruptionn Maintain stable immune function off ART May or may not be virus free

8 Functional Cure of the Berlin Patient with chemotherapy and HSCT Hutter G, et al NEJM 2009,360: Hutter G and Thiel. AIDS 2011,25:273-4.

9 Possible Reasons for Non-detectable HIV off ART (remission) in the Berlin Patient Long term ART had reduced HIV burden Chemotherapy removed infected cells in patient Transplanted cells protected from HIV infection due to CCR5 delta 32 mutation Unrelated donor cells contributed to a GVH-like reaction further clearance of latently infected cells Generation of host protective immunity Combination of above Did they just get lucky?

10 Eradication Not So Simple Latently-infected resting memory CD4 + T cells persist as viral reservoirs despite years of ART Virus RNA detected in blood and other lymphoid tissues of patients suppressed for >10 years t 1/2 of latently-infected resting memory CD4 + T cells is ~ 44 months making their elimination possible in ~70 years (if fully compliant with no breakthrough)

11 Interventions to possibly eliminate long-lived HIV-infected cells Block ongoing, low level HIV replication ART, anti-microbial, anti-inflammatory, anticoagulants, antifibrotics, anti-aging drugs Activate HIV-1 expression from latently-infected CD4+ memory T-cells, Kick and Kill Assumes this is the key reservoir Assumes activation is lethal for latently-infected cell Selectively kill cells expressing HIV-1 Generate antibodies or CI to HIV infected cells Non-selectively kill all or most cells which harbor HIV with chemo-rt and allow host immune system to mop up any residual reservoir cells (may require further immune activation or HIV vaccination) Protect new cells from HIV infection

12 Starting ART VERY Early Mississippi baby: started ART within 31 hours; stopped ART at 18 mos; now thought cured at age 41 mos (23 months off ART) Undetectable RNA; Trace HIV-1 DNA detectable Persaud NEJM 2013; 369; 1828 Long Beach baby: started ART within 4 hours; +HIV DNA PCR (4 hrs) and +HIV RNA (36 hrs); +CSF HIV PCR (day 6); no HIV RNA; no proviral HIV DNA after 6 days; now age 9 months on ART Persaud CROI 2014 #75LB

13 HIV-1 Reservoirs Reduced in HIV-Positive Children With Early ART and Viral Control 144 perinatally HIV-infected pts with long-term (median: 10.2 yrs) virologic suppression on ART Higher proviral burden with increasing age at virologic suppression In perinatally infected baby treated early (at 4 hrs of age) with triple ART, noninduced proviral genomes detected by PCR at 1 mo but not at 3 mos of age Proviral Reservoir Size by Age of Virologic Control Age, yr < 1 (n = 14) 1-5 (n = 53) > 5 (n =77) Median copies HIV DNA/ 10 6 PBMCs (IQR) 4.2 ( ) 19.4 ( ) 70.7 ( )* *P <.001 compared with < 1 yr Persaud D, et al. CROI Abstract 72. Persaud D, et al. CROI Abstract 48LB.

14 Starting ART VERY Early Patient on PrEP Demonstration Project HIV- at screening but HIV+ HIV at baseline RNA 220 cps/ml TDF/FTC X 7d (RNA 120), then cart (<40 after 32d) VL decreased <40 cps/ml by 32 days All other HIV RNA/ and DNA tests on PBMCs and subsets negative, including cell-associated and colon bx ART interruption planned after one year Hatano CROI 2014 #397LB

15 Kick and Kill Strategies Epigenetic inducers/inhibitors HDAC inhibitors: Voriniostat, romidepsin Proteosome inhibitors: Bortezomib DNA methylation inhibitors: Azacytidine, decitabine HAT and DNMT inhibitor: Flavonoids, hydralazine oxazolines, isoxazolines Non-specific activators Prostratin, Valproate, Disulfiram Protein kinase C activators: Bryostatin (+/- nanoparticles) Immune induction/activation IL-2, IL-7, IL-11 Anti-CD3 antibodies (OKT3) Immune-toxin conjugates

16 Multiple doses of vorinostat Margolis CROI 2014, # 435 LB

17 Therapeutic Interventions in Development Chemokine receptor inhibitors: maraviroc, TB-652 Anti-infective therapy: CMV, EBV, HSV, HCV/HBV Microbial translocation: sevelamer, colostrum, rifaximin Enhance T cell renewal: Growth Hormone, IL-7 Anti-fibrotic drugs: pirfenidone, ACEi, ARBs, KGF Anti-aging: caloric restriction, vit. D, omega-3 fatty acids, rapamycin, diet, exercise Anti-inflammatory drugs: Chloroquine, Hydroxychloroquine Minocycline NSAIDs (COX-2i, aspirin) Statins Methotrexate Anakinra (IL-1Ra) Thalidomide, lenalidomide, pentoxyfylline Biologics (TNFi, IL-6i, anti-ifna, anti-pd1, anti-pdl1, JAKi, IDOi, Casp-1i) Anti-coagulants: warfarin, dabigatran, aspirin, clopidogrel Immune enhancers: HIV vaccines and HIV antibodies Combination therapy may be necessary

18 HIV DNA (copies/10 6 PBMC) CD4 (cells/mm 3 ) HIV DNA (copies/10 6 PBMC) CD4 (cells/mm 3 ) Reduced HIV Reservoir after Allogeneic Stem Cell Transplant Patient A: PBMC DNA CD4 Count HCST 100% Donor Lymphocyte Chimerism 4.3 Years Post-HSCT (<0.07 copies/10 6 ) Post HSCT (days) Post HSCT (days) Patient B: PBMC DNA CD4 Count 100% Donor Lymphocyte Chimerism 2.6 Years Post-HSCT (<0.04 copies/10 6 ) HCST Post HSCT (days) Post HSCT (days). Henrich T, et al. 7 th IAS Conference. Kuala Lumpur, Abstract WeLBA05.

19 Allogeneic Stem Cell Transplant Patient A: No HIV RNA after 15 weeks off ART Rapid relapse of HIV after 32 weeks Developed acute HIV symptoms Genetic testing showed single/same HIV genotype Patient B: No HIV RNA after 7 weeks off ART Rapid relapse of HIV after 12 weeks Developed acute HIV symptoms Genetic testing showed single/same HIV genotype Henrich T et al CROI 2014, Boston, MA

20 Gene Therapy Definition Introduction of a gene transfer product into cells to produce a therapeutic benefit

21 Anti-HIV-1 Gene Therapy Recombinant T-cell receptor (CD4 zeta) Transdominant proteins (Rev M10, Trev, C46) Intracellular antibodies and RNA decoys Antisense (tat, RevM10, env ViRxsys) Ribozymes (U5 hairpin, Rz2 hammerhead) dsrna (RNAi, sirna) RNA aptamer (small RNA antagonists of protein function) Zinc finger nucleases (CCR5 directed, SB-728) David Baltimore, PhD Intracellular Immunization

22 Anti-HIV-1 Gene Therapy Recombinant T-cell receptor (CD4 zeta) Transdominant proteins (Rev M10, Trev, C46) Intracellular antibodies and RNA decoys Antisense (tat, RevM10, env ViRxsys) Ribozymes (U5 hairpin, Rz2 hammerhead) dsrna (RNAi, sirna) RNA aptamer (small RNA antagonists of protein function) Zinc finger nucleases (CCR5 directed, SB-728) David Baltimore, PhD Intracellular Immunization

23 Gene Therapy Targets CCR5r inhibitors TAR or RRE decoys Fusion inhibitors Zn Finger Nuclease Ribozymes Antisense shrnas Aptimers Rossi, June, Kohn. Nat Biotech 25:1444, 2007

24 Rationale for Use of Gene Transfer Approach in HIV Infection Intracellular gene therapy works without medications that can effect other organ systems Gene therapy may have long lasting effects and can be self perpetuating if placed in appropriate stem cells Gene modification may inhibit HIV replication and/or protect uninfected cells May be effective against multiple clades and drug resistant strains of HIV May be less prone to development of resistance Progeny of a small number of gene modified cells may have a survival advantage and persists for long time

25 Challenges in Gene Therapy Technical Challenges Selecting gene target(s) and inhibitory mechanisms Getting good transduction efficiency Getting good engraftment of transduced cells Conditioning regimen or transduction approach Apheresis and large cell processing/storage/shipping Monitoring cell marking and trafficking Scaleability and cost Subject Challenges Selecting appropriate patient population Identifying and recruiting subjects Long term safety follow up

26 Preliminary Results from 220 HIV+ treated with gene therapy HSC and T cells successfully harvested and gene modified In absence of conditioning, gene marking ranges from 0.01% to 0.38% (May need only 10%) No toxic effects or major side effects seen from procedure Encouraging early results for VL and CD4+ Tcell counts and decrease in inflammatory markers Selective advantage of gene modified cells demonstrated in blood, bone marrow and GALT Myeloablation or conditioning allows greater level of engraftment and gene marking (DiGusto et al, 2010) Cavazzana-Calvo, CROI 2013, Atlanta, abst 124

27 Zinc Finger Nuclease Studies

28 Zinc Finger Nucleases (ZFNs) Designer Restriction Enzyme ZFP D32 mutation DNA ZFP CCR5 ZFN modification Site 165 Heterodimer comprised of 2 domains - Nuclease domain of FokI restriction enzyme - Zinc finger protein provides DNA binding specificity; targets 12 nucleotide each ZFN cleavage results in a double stranded DNA break - Non-homologous end-joining repair leads to permanent CCR5 gene modification Delivered with a non-integrating, replication-deficient, chimeric adenoviral 5/35 vector Approximately 16-39% (mean = 23%) of the CCR5 modifications contain a CTGAT duplication (Pentamer)

29 SB-728-T GMP Manufacturing Process: Autologous ZFN CCR5-Disrupted CD4+ T-cells Leukapheresis Monocyte and CD8+ T cell depletion Enriched CD4+ Activate with Anti-CD3/28 beads Adenoviral SB-728 transduction and CCR5 disruption of CD4 cells Expansion in WAVE Infuse Cryopreserve cell product (SB-728-T) ~25% CCR5 modification

30 SB-728-T Increases CD4 T-cell Counts in Peripheral Blood after Infusion Cohort 1 Cohort 2 Cohort 3 Change in CD4 T-Cell Count from Baseline (per L) Median Cells Days 30

31 SB Treatment Interruption Subject 102 Viral Load Estimated CCR5 Modified CD4 T-Cells Treatment Interruption Log Viral Load (Copies/mL) Estimated CCR5 Modified CD4 T-Cells (% of CD4 Cells) Day 7 Month 3 Month 6 Month 9 Month 12 WEEK 2 WEEK 4 WEEK 6 WEEK 7 Time WEEK 8 WEEK 10 WEEK 12 WEEK 14 WEEK 16 WEEK 20 WEEK 24 WEEK 28 Mitsuyasu, ICAAC,

32 Gene Modified Stem Cells

33 Hematopoiesis Myeloid stem cell CD34 + progenitor cell Lymphoid stem cell Erythroid progenitor Megakaryoblast Eosinophil progenitor Basophil progenitor Myelomonocytic progenitor B progenitor T progenitor Thymocyte Monocyte Megakaryocyte Red blood cells Platelets Eosinophil Basophil Neutrophil Macrophage B cell CD8 + T cell CD4 + T cell

34 Cal-1 Clinical Trial Sponsored by Calimmune, Inc. California Institute for Regenerative Medicine Conducted at UCLA CARE Center and Quest in SF

35 Engineering Protection Anti-HIV Gene A A A CD4+ cells A T lymphocyte development Autologous Hematopoietic Stem Cells A A A Macrophages Myeloid development Delivery to Hematopoietic Stem/Progenitor Cells for long-term protection Delivery to CD4+ T Cells for short- to medium-term protection Intracellular immunization: Progressive population of immune system with cells protected against HIV

36 Investigational Product Cal-1 is a self-inactivating lentiviral vector that contains two active anti-hiv agents Sh5 is a short hairpin RNA against the HIV-1 co-receptor CCR5 C46 is a membrane-anchored C-peptide derived from the HIV-1 envelope glycoprotein gp41 Active against both R5- and X4- strains of HIV Two points of inhibition for R5-tropic HIV-1 Mitigates against resistance of HIV 3 7

37 Cell Processing Overview 1. Apheresis Small volume apheresis Standard volume apheresis G-CSF HIV-Infected individual HSPC tn Busulfan to create bone marrow space Infuse cells T tn 5. Autologous transplant of genetically modified cells CD4 + cell isolation with CliniMacs CD34 + cell isolation with CliniMacs Isolated CD34 + Transduction with Cal-1 4. Harvest of transduced CD4+ and CD34+ cells 2. Cell isolation 3. Lentiviral Transduction Isolated CD4 +

38 Directions for moving forward Improve gene product effects against HIV multiple targets and potent constructs Better, safer transduction of cells (transduction >90%) Automate transduction procedure Improve engraftment of marked cells (conditioning) Multiple cell types (HSC and T cells M/M and others) Determine the best means of applying selection pressure for proliferation and differentiation of cells (off ART) Determine distribution and functionality of gene marked cells (in blood, BM, GALT, LN) Combine with other anti-hiv immune strategies (e.g. therapeutic vaccines) May be part of the Functional Cure of HIV

39 Curing HIV is: 0% A.Impossible 0% 0% 0% 0% 0% B. Requires removing all HIV from the host C. Means controlling HIV without antiretroviral therapy D.Requires high dose chemotherapy E. Is only possible in newborns F. B + D

40 Possible approaches to curing HIV are: 0% 0% 0% 0% 0% A. Continuing ART for > 70 yrs with no missed doses B. Requires inducing HIV from latent reservoirs C. Only occurs with bone marrow or stem cell transplants D. Will likely require multiple strategies E. Means the person cannot be infected again with HIV

41 Thank You Questions and Comments