Curing HIV:

Coping with Hope

Ronald Mitsuyasu, MD Professor of Medicine

UCLA Center for Clinical AIDS Research

and Education (CARE Center)

Curing HIV is:

0%

0%

0%

0%

0%

0% A. Impossible

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

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

Antiretroviral Drugs 2014 (28)

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)

Protease Inhibitors (10)

• saquinavir (SQV)

• ritonavir (RTV)

• indinavir (IDV)

• nelfinavir (NFV)

• amprenavir (APV)

• lopinavir/r (LPV/r)

• fosamprenavir (FPV)

• atazanavir (ATV)

• tipranavir (TPV)

• darunavir (DRV)

Reverse Transcriptase Inhibitors(13)

Integrase Inhibitor (3) •raltegravir (RAL)

•elvitegravir (ELV)

•dolutegravir (DTG)

Fusion Inhibitor •Infuvirtide (Fuzeon)

Entry Inhibitor (CCR5) •maraviroc (MVC)

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

Major Problem with ART

Federal Resources / Policies/Issues : HIV/AIDS Care Continuum

http://aids.gov/federal-resources/index.html

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

Functional Cure of the “Berlin Patient”

with chemotherapy and HSCT

Hutter G, et al NEJM 2009,360:692-698.

Hutter G and Thiel. AIDS 2011,25:273-4.

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?

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

t1/2 of latently-infected resting memory

CD4+ T cells is ~ 44 months making their

elimination possible in ~70 years (if fully

compliant with no breakthrough)

Interventions to possibly eliminate

long-lived HIV-infected cells

Block ongoing, low level HIV replication

• ART, anti-microbial, anti-inflammatory, anti-coagulants, 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

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

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

Persaud D, et al. CROI 2014. Abstract 72.

Persaud D, et al. CROI 2013. Abstract 48LB.

Proviral Reservoir Size by Age of

Virologic Control

Age, yr Median copies HIV DNA/ 106 PBMCs (IQR)

< 1

(n = 14)

4.2 (2.6-8.6)

1-5

(n = 53)

19.4 (5.5-99.8)

> 5

(n =77)

70.7 (23.2-70.7)*

*P < .001 compared with < 1 yr

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 (

“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

Multiple doses of vorinostat

Margolis CROI 2014, # 435 LB

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

Therapeutic Interventions in Development

Combination therapy may be necessary

Reduced HIV Reservoir after Allogeneic Stem Cell Transplant

Henrich T, et al. 7th IAS Conference. Kuala Lumpur, 2013. Abstract WeLBA05 .

.

Patient A: PBMC DNA CD4 Count

Post HSCT (days)

0 200 400 600 800 1000 1200 1400 1600

HIV

DN

A

(co

pie

s/1

06 P

BM

C)

Post HSCT (days)

0 200 400 600 800 1000 1200 1400 1600

CD

4 (

ce

lls

/mm

3) 4.3 Years

Post-HSCT (

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

Gene Therapy Definition

Introduction of a gene

transfer product into cells

to produce a therapeutic

benefit

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”

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”

Gene Therapy Targets

Rossi, June, Kohn. Nat Biotech 25:1444, 2007

Zn Finger Nuclease

CCR5r inhibitors

Fusion inhibitors

Antisense Ribozymes

TAR or RRE decoys

shRNAs

Aptimers

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

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

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

Zinc Finger Nuclease Studies

ZFP

ZFP

Zinc Finger Nucleases (ZFNs) “Designer Restriction Enzyme”

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)

DNA

CCR5

ZFN modification

Site 165

D32 mutation

SB-728-T GMP Manufacturing Process:

Autologous ZFN CCR5-Disrupted CD4+ T-cells

Leukapheresis

Enriched

CD4+

Adenoviral SB-728

transduction and

CCR5 disruption

of CD4 cells

Cryopreserve

cell product

(SB-728-T)

~25% CCR5

modification

Infuse

Activate with

Anti-CD3/28

beads

Monocyte and

CD8+ T cell

depletion

Expansion in WAVE

30

SB-728-T Increases CD4 T-cell Counts

in Peripheral Blood after Infusion

102 103 104

201 203 302

303304305.

Median

Cohort 1 Cohort 2 Cohort 3

Days

0 30 60 90 120 150 180 210 240 270 300 330 360

Ch

an

ge

in

CD

4 T

-Ce

ll C

ou

nt

fro

m B

as

eli

ne

(pe

r L

)

-200

0

200

400

600

800

1000

1200

100 Cells

31

SB-728-0902

Treatment Interruption – Subject 102

Treatment Interruption

Time

0

Day

7

Month

3

Month

6

Month

9M

onth

12

WE

EK

2

WE

EK

4

WE

EK

6

WE

EK

7

WE

EK

8W

EE

K 1

0W

EE

K 1

2W

EE

K 1

4W

EE

K 1

6W

EE

K 2

0W

EE

K 2

4W

EE

K 2

8

Lo

g V

iral

Lo

ad

(C

op

ies/m

L)

101

102

103

104

105

106

Esti

mate

d C

CR

5 M

od

ifie

d C

D4 T

-Cell

s

(% o

f C

D4

Cells

)

0

2

4

6

8

Viral Load

Estimated CCR5 Modified CD4 T-Cells

Mitsuyasu, ICAAC, 2010

Gene Modified Stem Cells

Neutrophil

CD34+

progenitor

cell

Myeloid stem cell

T progenitor

Thymocyte

CD4+

T cell B cell

Megakaryoblast Erythroid

progenitor

Eosinophil

progenitor

Platelets Red blood cells

Myelomonocytic

progenitor

Basophil

progenitor

Basophil Eosinophil

Lymphoid stem cell

Megakaryocyte

B progenitor

Macrophage

Monocyte

CD8+ T cell

Hematopoiesis

Cal-1 Clinical Trial

Sponsored by Calimmune, Inc.

California Institute for Regenerative Medicine

Conducted at

UCLA CARE Center and Quest in SF

Anti-HIV Gene

Autologous

Hematopoietic Stem Cells

A

A

Macrophages A

CD4+ cells

T lymphocyte development

A

Myeloid development

A A A

Intracellular immunization:

Progressive population of immune system

with cells protected against HIV

Engineering Protection

• Delivery to Hematopoietic Stem/Progenitor Cells for long-term

protection

• Delivery to CD4+ T Cells for short- to medium-term protection

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

3

7

Investigational Product

•Active against both R5- and X4- strains of HIV •Two points of inhibition for R5-tropic HIV-1 •Mitigates against resistance of HIV

Cell Processing Overview

HIV-Infected individual

Standard volume

apheresis

G-CSF

Small volume

apheresis

CD34+ cell isolation with

CliniMacs

CD4+cell isolation with

CliniMacs

Infuse cells

Isolated CD4+

Transduction with Cal-1

HSPCtn Ttn

Isolated CD34+

1. Apheresis

2. Cell isolation 3. Lentiviral Transduction

5. Autologous transplant of genetically modified cells

4. Harvest of transduced CD4+ and CD34+ cells

Busulfan to create bone marrow “space”

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”

Curing HIV is:

0%

0%

0%

0%

0%

0% A. Impossible

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

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

Thank You

Questions and Comments