Kamel Khalili, Ph.D.Laura H. Carnell Professor and Chair

Department of Neuroscience

Director

Center for Neurovirology &

Comprehensive NeuroAIDS Center

Co-Director, Center for Translational AIDS Research

Lewis Katz School of Medicine at Temple University

Philadelphia, Pennsylvania

Gene Editing Strategy for Eliminating

HIV-1/AIDS

Workshop on HIV&Aging

September 2016

The Aging Face of AIDS

cART

Neurocognitive disorders

Depression

Cardiomyopathy

Dislipidemia

Renal Disease

Other comorbidities

An Integrated Copy of HIV-1 DNA in

Host Chromosome

Wong et al, 2011 PLoS One

The CRISPR/Cas9 Gene Editing System

Schematic of CRISPR/Cas9 HIV-1 DNA Editing

Eradication of HIV-1 DNA in Latently

Infected T-Cells

A

B

Elimination of the Proviral DNA is Shown by

Ultra Deep Whole Genome Sequencing

Elimination of Proviral DNA from

Host Chromosomes

A

B

Can Cured T-cells Be Infected with HIV-1?

Patient Derived Primary PBMCs and CD4+ T-cells

Cas9 Delivery by AAV

In vivo editing of HIV-1 by CRISPR:

small animal models

• Tg26 transgenic mice

transgenic mice containing entire HIV pNL4-3 (partial Dgag/pol)

HIV transgenic rats

transgenic mice containing entire HIV pNL4-3 (partial Dgag/pol)

• EcoHIV infected mice

chimeric HIV containing MuLV envelope and Luciferase reporter –

virus is replication competent in mice

• BLT humanized mice

circulating human-derived PBMCs – supports HIV replication in vivo

Excision of HIV-1 DNA by rAAV9:saCas9/gRNA in Tg26 MEF

Eradication of HIV-1 in An Animal Model:

In Vivo Study

B

Elimination of Segments of the Integrated

HIV-1 DNA from Rat Blood Cells

Bioluminescence Imaging Analysis

TA Cloning and Sanger Sequencing

Intraperiotoneal Infection of BLT Mice

with HIV-NL-BAL-eLuc

Dorsal D2 p.i. D5 p.i. D7 p.i. D14 p.i.Ventral

3.1 X 104 TCIU

D21 p.i. D28 p.i.

Kidney

Lung

Liver

Human thymic

organoidSpleen

Kinetics of Infection

A

B

B7M2 Mice Intraperiotoneally Inoculated

With HIV-BAL-eLuc

D86D65

D16 D22 D30 D37

D44

D2

D52

1 x 106 TCIU

AAV-Cas9 (IV)

B7M2 Intravenously Inoculated

with AAV/Cas9 (20µl).

D93 p.i.

T758/T363

(35 cycles)

1500

500

B10M4 Intraperitoneally Inoculated

with HIV-BaL-eLuc(1x105 TCIU)

D3 p.i. D7 p.i. D14 p.i. D21 p.i.

ART (TFV/FTC/RAL)

D28 p.i. / D5 p.a D35 p.i. / D12 p.a D63 p.i. / D25 p.w.

HIV rebound

D42 p.i. / D4 p.a

ART Withdrawal

p.i., Post-inoculation; p.a., Post-antiretroviral therapy (ART); p.w., Post-withdrawal of ART

Acknowledgments

Supported by grants awarded by NIH to KK, BW, JK, RB, and HG.

Infrastructure support: Comprehensive NeuroAIDS Center (CNAC)

Funded by NIMH (P30MH092177)

Temple University (KSOM)

Rafal Kaminski, Ph.D.

Hassen Wollebo, Ph.D.

Yilan Chen, M.D., Ph.D.

Wenhui Hu, M.D., Ph.D.

Chaoran Yin, M.D.

Fang Li

Yonggang Zhang, M.D., Ph.D.

Tracy Fischer, Ph.D.

Ellen Tedaldi, M.D.

Jeffrey Jacobson, M.D.

Julian Salkind

Ramona Bella

Pasquale Ferrante

(University of Milan)

Drexel University (DUCOM)

Brian Wigdahl, Ph.D.

and his team

George Mason University

Fatah Kashanchi, Ph.D.

and his team

Florida International University

Madhavan Nair, Ph.D.

and his team.

University of Nebraska (UNMC)

Howard Gendelman, M.D.

and his team

Case Western Reserve

University (CWRU)

Jonathan Karn, Ph.D.

and his team

University of South Carolina

Rosemarie Booze, Ph.D.

and her team

Disclosures

Kamel Khalili is a board member, scientific advisor, and holds equity in Excision

Biotherapeutics, a biotech start-up who has licensed the viral gene editing technology

from Temple University for commercial development and clinical trials.

Funding