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
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
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?
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