Gene Therapy- Past, Present and Future

Mark A. Kay MD, PhDDennis Farrey Family Professor

Departments of Pediatrics and GeneticsStanford University

Definition of gene therapy

Gene therapy is the introduction of nucleic acids (e.g. DNA/genes) into somatic cells of the body to correct or prevent a pathological process

Think of DNA as a class of pharmaceuticals

•Fix a gene change the DNA-mutation repair

•Silence a gene-from a pathogen,gain of function mutation

What Do We Want to Accomplish?

•Add a gene-restore a missing gene function or supply an RNA or protein that has pharmacological effect

Vector Categories

Viral-Based-Manipulating Natural

Existing Viruses to Transfer

Therapeutic Genes

Non-Viral- DNA Molecules Delivered

as Naked DNA, Lipids, or Polymers

Which vectors are in use?

What diseases are being targeted?

Potential factors limiting vector efficacy

Adeno-associated viruses

Discovered in mid 1960s as a contaminant

Family: ParvoviridaeSubfamily: ParvovirinaeGenus: Dependoparvovirus

AAV Vectors

CapRep DNA

AAV

ITR

Therapeutic geneControlelement

Gene Cassette

DNA

AAV Vectors

ITR ITR

ITR

AAV vector production strategies: Helper virus-free system

Therapeutic gene

ITRITR

pVector

CapRep

pHLP

pladenoE2A

E4VA

CaPO4

Transfection in 293 cells

Vector recovery

Alternative Production Methods

Purification Schemes

• Various Physical Separation methods

• Vector purity (e.g. advential agents, DNA contamination)

• Full to Empty Capsid Ratio

• Capsid Composition

rAAV-mediated transgene expression in vivo

AAV-EF1a-hFIX to C57BL/6 mice

1000

100

10

0 2 4 6 8 10

hF

IX (

ng

/ml)

Months

Snyder et al., Nat. Genetics 1997

Nature Genetics, 1997

First Hemophilia A dog

Dogs are treatedDog colonies are University of North Carolina at Chapel Hill

Dogs treated with AAV-FIX vectors

Expression for > 7 years

Hemophilia B dog

Kenneth M. Brinkhous at the University of North Carolina at Chapel Hill

Biotech - Academic Collaboration

• The ultimate goals are the same• The acute similarities & differences

• Disclosures• IP restrictions• Survival –publish vs perish

shareholders

First systemic administration of an AAV vector into a human

Manno et al., Nature Medicine 2006

Successful transduction of liver in hemophilia by AAV-Factor IX and

limitations imposed by the immune host response hemophilia

• Therapeutic hFIX was demonstrated in a human

• Unlike all animal models expression in humans was temporary

• This limitation was likely related to a cell mediated immune directed against hepatocytes containing capsid particles

• No matter how good the animal models one cannot predict the outcome in humans until you try it in people

Manno et al., Nature Medicine 2006

Surprises along the way

• Jessie Gelsinger’s Death

• Seminal Fluid PCR detection of vector DNA genomes--- how to establish significance

• Unexplained Transaminitis

Pseudotyping recombinant vector genomes

• AAV-2 is prototype vector-isolated from humans

• Most of the population exposed (e.g. immunity)

• Cell-mediated immune responses

• Small number of amino acid changes can have profound effects

• on the transduction parameters (immunity, efficiency, cell type)

AAV2AAV1 AAV3

AAV4 AAV5 AAV6

Nathwani et al., New England Journal of Medicine 2011

AAV-2/8 Gene Therapy for Hemophilia B

AAV-8 is not an optimal vector

for human gene transfer

• Some humans pre-existing immunityinhibiting any gene transfer

• Dose response in human is >10x less than expected based on animal studies

Various AAV-clinical trials for

Hemophilia B ongoing

• Additional patients with better dosing

• What about patients with pre-existing immunity?

• What happens if expression wanes over time?

• How long will it last if treated at birth?

How does one predict clinical outcomes from animal studies?

Which animals models are most predictive?

Reconstitution of mouse liver with human hepatocytes

AV shuffle library screening in FRG mice

Succinylacetone

Hpd-/-NTBC

HT1 Fah-/-

Tyrosine

P-hydroxy-phenylpyruvate

AcetoacetateFumarate

Fumarylacetoacetate (FAA)

Maleylacetoacetate (FAA)

Homogentistisic acid (HGA)

Azuma et al., Nature Biotechnology 2007

Molecular

shuffling and

evolution of new

viruses

Kay, M.A. Nature Reviews Genetics 2011

AAV shuffle library selection in Tissue Culture Cells

TC cells

Harvest supernatanton day 3 post infection

AAV library (MOI range)hAd5 (fixed predetermined MOI)

Western Blot analysis

Select dilution with the lowestexpression of AAV proteins

VP1VP2VP3

Library MOI

•Vector DNA extraction

•PCR amplification of CAP gene

• TOPO subcloning

• Sequencing

Human IVIG negative selection

Library screening in mice with humanized liver

Lisowski et al., Nature 2014

Vectorization of AAV capsids obtained from in vivo screen

Lisowski et al., Nature 2014

Limitations to conventional rAAVapproaches

• Loss of transgene expression in growing or dividing cells

• Risk of insertional mutagenesis

• Problems with re-administration

• Genome size

• Pre-existing immunity

• Cell-mediated immunity

Genome editing

without nucleases

ITRhF92A

5’ homology arm to Alb 3’ homology arm to Alb

… …Wild-type Alblocus

Targeted Alblocus ……

RNA: Fused mRNA

Protein: +

Stop

P hF9

Alb

Alb 2A

DNA:

AAV8 capsid

hF92A

hF92A

Promoterless Gene Targeting Without Nucleases

Barzel et al., Nature 2014

Promoterless Gene Targeting for hFIX

Improved Human Capsid + Gene Ride

• Treat non-quiescent tissues including neonatal

liver. No retreatment with rAAV required

• Abrogates hepatic genotoxicity (hepatocellular carcinoma)

• No genotoxicity or immunogenicity associated with nucleases

• Endogenous gene regulation

Where do we go from here?