Paula CannonUniversity of Southern California

Los Angeles, CA, USA

CRISPR/Cas and Gene Editing for HIV

Disclosures relevant to this talk

• Scientific advisor, Sangamo Therapeutics

• Consultant, MilliporeSigma

• Why HIV?

• Gene editing basics

• Opportunities against HIV

• The next 20 years

Integrated latent but re-activatable HIV prevents cure by ART

Gene editing tools recognize specific DNA sequences

CRISPR-Cas9• CRISPR is a homologous guide RNA

• Cas9 is a an endonuclease

Zinc finger nucleases• DNA-binding peptides in arrays

• Linked to a non-specific endonuclease

guide RNA

Cas9

DNA break repair can disrupt a gene

InDels

NHEJ

InDels

NHEJ

DNA break repair can disrupt a gene – or an integrated provirus

Homology-directed repair increases gene editing possibilities

InDels

HDR

Gene edit

+ homologous

DNA template

Site-specific

gene addition

NHEJ

‘Dead Cas9’ fusions modulate gene expression without DNA breaks

Transcription on Transcription off Epigenetic modifications

Dead Cas9

- just DNA binding

Mechanism Potential applications

NHEJ indels • Gene knockout to confer HIV resistance (CCR5)

• Disrupt integrated provirus

HDR editing

& insertions

• Site-specific insertions of anti-HIV genes

• Engineer protective anti-HIV alleles

• Engineer immune effectors to recognize HIV or

HIV-infected cells

dCas9 transcription

regulation

• Modulate host genes to disrupt HIV replication

• Shock, block or lock latent HIV

Hematopoietic stem cell transplants from CCR5D32 donors: 3 cures?

Berlin Patient1 London Patient2 Dusseldorf Patient3

Malignancy AML HL AML

ART post-HSCT none 16 mths 66 mths

HIV remission > 10 yrs 18 mths 3 mths

1. Hutter NEJM 2009

2. Gupta Nature 2019

3. Jensen CROI 2019

Can gene editing recapitulate CCR5D32 HSC transplantation?

• Patients receive 100% CCR5-negative donor HSC

• Cancer therapies (chemo/radio/immune) deplete the latent reservoir

• Graft versus host attack by donor cells further depletes the reservoir

Allogeneic HSC transplant

Autologous HSC engineering

Holt, Nat Biotech 2010

Disrupting CCR5 gene in HSC using zinc finger nucleases

Humanized mice

Human CD4 T cells in blood

HIV in mouse blood

sCurrent clinical trials targeting CCR5

Autologous T cells edited with ZFNs• U Penn, UCSF, UCLA, Sangamo Therapeutics • Tebas et al., NEJM 2014

Autologous HSC edited with ZFNs• City of Hope, Sangamo Therapeutics

Edit donor HSC with CRISPR/Cas for hematologic malignancy• Affiliated Hospital to the Academy of Military Medicine, Beijing University

CCR5D32 donor HSC for hematologic malignancy• IciStem consortium

Mechanism Potential applications

NHEJ indels • Gene knockout to confer HIV resistance (CCR5)

• Disrupt integrated provirus

HDR editing

& insertions

• Site-specific insertions of anti-HIV genes

• Engineer protective anti-HIV alleles

• Engineer immune effectors to recognize HIV or

HIV-infected cells

dCas9 transcription

regulation

• Modulate host genes to disrupt HIV replication

• Shock, block or lock latent HIV

HDR

Site-specific addition of eCD4-Ig at CCR5

CCR5

Gardner Nature 2015

eCD4-Ig

eCD4-Ig CCR5 negative and

eCD4-Ig expressing

sCD4

Fc

CCR5 mimetic

CCR5

CD4eCD4-Ig

Site-specific addition of eCD4-Ig at CCR5

6 9 12 150

200

400

600

800

1000

Weeks post transplant

eC

D4Ig

G in

pla

sm

a

(ng

/mL

)

eCD4IgG in plasma

780782

784

793

Edited

R5-eCD4IgG

found dead on wk16

(CD45+ levels

wk12 = 8%

wk15 = 57%)

eCD4-Ig edited HSC

Challenged X4 HIV

= resistant

HDR

CCR5

eCD4-Ig

eCD4-Ig CCR5 negative and

eCD4-Ig expressing

Mechanism Potential applications

NHEJ indels • Gene knockout to confer HIV resistance (CCR5)

• Disrupt integrated provirus

HDR editing

& insertions

• Site-specific insertions of anti-HIV genes

• Engineer protective anti-HIV alleles

• Engineer immune effectors to recognize HIV or

HIV-infected cells

dCas9 transcription

regulation

• Modulate host genes to disrupt HIV replication

• Shock, block or lock latent HIV

• Protective alleles/SNPs – CCR5D32, HLA-B57

• Clues from primate orthologs of restriction factors

Candidate genes to edit?

Restriction factors – intracellular anti-HIV defenses

Restriction factor

HIV antagonist

TRIM5a

R332/335G

Rhesus macaque

Sooty MangabeyTRIM-Cyp 0 10 20 30 40

106

107

108

109

1010

Days post infection

To

tal H

IV c

op

ies

Viral load, at-site editing, +IFN

1-21-31-45

GFP

Ctrl

TRIM-CypR332/5G

HIV levels in humanized mice

• Recognizes HIV cores (terminal SPRY domain)

• Disrupts HIV uncoating and triggers NFkB

• HIV-1 cores protected by a cyclophilin A coat

Human

SPRY

Mechanism Potential applications

NHEJ indels • Gene knockout to confer HIV resistance (CCR5)

• Disrupt integrated provirus

HDR editing

& insertions

• Site-specific insertions of anti-HIV genes

• Engineer protective anti-HIV alleles

• Engineer immune effectors to recognize HIV

or HIV-infected cells

dCas9 transcription

regulation

• Modulate host genes to disrupt HIV replication

• Shock, block or lock latent HIV

• TCRs recognize peptides

presented by MHC-I

• CARs redirect to other cell

surface antigens, eg using

single chain antibodies

Chimeric Antigen Receptors redirect CD8 T cell killing

CD8 T cell CD4 ectodomain

recognizes Env

Gene editing can improve CAR T cells

• HDR editing can insert CAR cassette at a defined locus, including native TCR

• PD-1 gene knockout prevents T cell exhaustion

Towards universal ‘off the shelf’ reagents:

• b2M / HLA gene knockout prevents rejection

of allogeneic CAR T cells by host

• TCR gene knockout prevents unwanted

graft vs. host reactions

• Strategy is double cut drop-out and

replace by VDJ cassette from bnAb PG9

Voss JE, Elife 2019 Jan 17;8

Gene editing the immunoglobulin locus to express bnAbs

• Edited human B cells bind HIV Env

Mechanism Potential applications

NHEJ indels • Gene knockout to confer HIV resistance (CCR5)

• Disrupt integrated provirus

HDR editing

& insertions

• Site-specific insertions of anti-HIV genes

• Engineer protective anti-HIV alleles

• Engineer immune effectors to recognize HIV or

HIV-infected cells

dCas9 transcription

regulation

• Modulate host genes to disrupt HIV replication

• Shock, block or lock latent HIV

LTR-targeted nucleases to disrupt integrated proviruses

Llewellyn, J. Virol. 2019

Relative HIV induction from

latently infected splenocytes

Mock Ctrl.

nuclease

LTR

nuclease

LTR LTR

• Why HIV?

• Gene editing basics

• Opportunities against HIV

• The next 20 years

– and some reality checks

Is this right for HIV?

- complex gene/cell therapies for a disease you can treat with drugs

What about safety?

- gene editing safety not yet established, limited clinical trials

Can gene editing be a one-time treatment?

- won’t have to worry about adherence, but monitoring is challenging

Public acceptance?

- CRISPR babies don’t help

CRISPR/Cas as a drug

• Several gene therapies are now approved

• Cancer CAR T cell therapies are driving innovation in manufacturing

• Delivery is the challenge:

- ex vivo, RNA, nanoparticles, viral vectors etc

• Pricing

- value

- up-front vs. subscription

- linked to outcome

ASGCT position paper. Mol Ther Dec 2018

Cannon Lab, USC Sangamo TherapeuticsAndreas Reik

Ed Rebar

Gary Lee

Jianbin Wang

Michael Holmes

The Scripps Research InstituteJames Voss

Dennis Burton

Kristina Tatiossian

Robert Clark

Chun Huang

Eduardo Seclen

Nick Llewellyn

Geoff Rogers

Camille Chen

Evan Lopez

Heidy Morales

Past members:

Danielle Krasner

Cathy Wang

Colin Exline

Nat Holt

U19 HL129902

R33 AI 110149

R01 DE 025167

R01 MH113457

Acknowledgments