Gene Transfer and Immunogenicity Branch

CTGTAC MeetingNovember 29, 2012

Andrew Byrnes, PhD, Senior Investigator, Branch ChiefCarolyn Wilson, PhD, Senior Investigator, CBER ADR

Suzanne Epstein, PhD, Senior Investigator, OCTGT ADRGraeme Price, PhD, Staff Fellow

Jakob Reiser, PhD, Senior Staff FellowWu Ou, MD, Staff Fellow

Cheng-Hong Wei, PhD, Visiting Scientist

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Overview of the Gene Transfer and Immunogenicity Branch

Examples of regulated productsGene therapy products

T cell products

Stem cell-derived products

Therapeutic vaccines

Xenotransplantation products

Mission relevance of researchImproving product safety and efficacy

Developing better preclinical models

Characterizing complex products

Addressing other FDA and HHS priorities:Pandemic influenza

Counter-bioterrorism

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Overview of the Gene Transfer and Immunogenicity Branch

Research topicsVirology and gene therapy

Gamma retroviruses

Lentiviral vectors

Adenoviral vectors

Influenza virus

Ebola virus

ImmunologyImmune regulation

Autoimmunity

Immune responses to viral and plasmid vectors

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Viral safety studies in xenotransplantation

Carolyn Wilson

Xenotransplantation raises public health concerns

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What are the viral and cellular determinants that allow PERV to

infect human cells?

Goals:1) Identify regions of viral envelope

important for human cell entry

2) Identify structural features of receptor for PERV-A required for viral infection

Major finding: Human cells express receptorsfor three classes of PERV

IMPLICATIONS FOR XENOTRANSPLANTATION:

Our data suggest that PERV-C envelopes may adapt to use the human

PERV-C receptor through mutation of a single residue

Argaw, et al, Journal of Virology, 20127

Identify structural features of receptor for PERV-A required for viral infection

PERV-A receptor = Riboflavin Transporter8

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Cross-linking suggests multimeric PERV-A receptor (huPAR2)

SIR

C

SIR

C-h

uPA

R2

SIR

C-h

uPA

R2

SIR

C-h

uPA

R2

BS3: - - 5 10 mM

Multimerizationrequired for infection?

Determine structural and functional attributes related to infectivity and multimerization

Library of huPAR-2

cDNAs with Cys-Ser mutations

Express in non-permissive cells

Initial Screen:•Receptor expression •Infectivity titer

Potential Impact of Studies: Improved understanding of cellular factors that influence human cell infection

If 1000-fold or greater decrease in titer:

Determine multimerization

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Universal influenza vaccines

Suzanne EpsteinGraeme Price

Influenza, the public health problem

High mortality from seasonal outbreaks, concern about pandemics.

Strain-matched vaccine can be delayed or insufficient

Work in this program: Universal influenza vaccines Cross-protection in animals by nucleoprotein (NP) and/or matrix 2

(M2) expressed by plasmid DNA or recombinant viral vectors. Investigation of possible cross-protection in humans

Relevance to CBER’s public health mission Center-wide priority on control of epidemic and pandemic influenza Gene therapy and tumor vaccines: Need to understand immune

responses to viral and plasmid vectors; therapeutic or interfering. Counter-bioterrorism: Control of emerging infections without

knowing which strain is coming

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Single dose mucosal emergency vaccine candidate protects mice against H5N1

Strong antibody and T cell responses induced, including locally in lungs. Protection against challenge with 10 LD50 of A/VN1203 (H5N1) 10 months post-immunization

Protection also seen as early as 2 weeks post-immunization

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Cross-protection by NP+M2 vaccinations in animal models

NP and/or M2 universal vaccine candidates protect mice against divergent influenza virus strains, including H1N1, H3N2, and H5N1.

In mice, intranasal rAd induces strong mucosal responses (IgA, lung T cells), strong protection against challenge.

Prime-boost vaccination to A/NP+M2 protects ferrets against H5N1 challenge, with reduction in nasal virus shedding.

Mouse model of transmission demonstrates that NP+M2 vaccination reduces spread of infection to contacts. Suggests vaccination could protect both recipients and the community.

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Human influenza surveillance study during the 2009 pandemic

Cross-protection in humans?

Did cross-reactive immune memory from past influenza exposures provide some protection against the 2009 pandemic strain?

Baseline sera and PBMC collected, donors monitored during fall 2009 pandemic wave. Those with influenza-like illness tested for virus.

Mild outbreak; few cases in cohort.

With Jack Gorski, Blood Center of Wisconsin

Analytical phase now in progress.

T cells, cytokines, antibodies

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Public health implications: Broad cross-protection for control of influenza

Broadly protective influenza vaccines could be used off the shelf early in an outbreak, when matched vaccines are not yet available, and could perhaps someday be used routinely.

Reduce illness, death, viral titers, spread of infection

With recovery from mild or asymptomatic infection, people would make antibodies to the strain circulating locally

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Immunoregulation and T cell tolerance induction

Cheng-Hong Wei

NOD

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Focus of research

• In vivo expansion of regulatory T cells (Tregs) prevents type 1 diabetes in mice

• Assays to measure the immunosuppressive activity of human MSCs

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Regulatory T cells (Treg cells)

• Foxp3+CD4+CD25+ regulatory T (Treg) cells are essential for the control of autoimmunity.

• Treg cells can suppress the proliferation and function of effector T cells.

• Therefore, Tregs are viewed as promising therapeutics for prevention/treatment of autoimmune diseases, transplant rejection

Major Findings (I)

1. A new combined regimen can significantly expand CD4+CD25+ regulatory T cells in vivo.

• Peptide + IL2 antibody complex + rapamycin

2. The expanded Tregs express a classical CD4+CD25+ Treg phenotype and are functionally suppressive both in vitro and in vivo.

3. Most importantly, the combined regimen significantly protects NOD mice against both spontaneous and induced type 1 diabetes.

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MSC = Multipotent Stromal Cells(or Mesenchymal Stem Cells)

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Major Findings (II)

1. Currently, there is a need for potency assays that measure the immunosuppressive activity of human MSCs.

2. In this work, we find that human MSCs can alter multiple aspects of clonal murine T cell activation, including:

• Proliferation

• Surface activation markers

• Cytokine production

• Transcription factors

3. Therefore, clonal murine T cells can be used to measure the immunosuppressive activity of human MSCs. This is a promising approach to measure product potency and to elucidate the mechanisms for immunosuppression

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Lentiviral vector safety and targeting

Jakob ReiserWu Ou

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Background

The goal of the Reiser lab is to develop safer HIV-1-based lentiviral vectors by:

• Directing vector integration to sites in the human genome that are devoid of proto-oncogene/tumor suppressor sequences (“safe harbor sites”)

• Narrowing the vector’s tissue tropism through targeted transduction

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Targeted lentiviral vector integration

• Ongoing approach: Use of integrase-defective lentiviral vectors bearing long homology arms to mediate homologous recombination (HR) at genomic “safe harbor” sites (such as the AAVS1 site)

• Future plans:– To improve HR frequencies using zinc finger

nickases– To use zinc finger recombinases to mediate

site-specific transgene integration

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Fusion protein

Cell-specific protein ligand (e.g. IL-13)Cell-specific RNA aptamer (e.g. anti-IL-13R2 aptamer)

Receptor (e.g. IL-13R2)

Targeted lentiviral vector transduction: General outline

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Targeted lentiviral vector transduction

• Future plans: – To further pursue IL-13R2-positive tumor

cells as a model for targeted vector delivery in vivo

– To design high-affinity RNA aptamers to allow high-efficiency targeting of IL-13R2-positive cells in vivo

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Adenovirus VectorBiodistribution and Toxicity

Andrew Byrnes

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Adenovirus:A popular vector in clinical trials

A large number of active adenovirus INDs:

~ 50 Ad gene therapies and oncolytic Ad vectors

~ 30 Ad-based vaccines

Two approved adenoviruses in China

Gendicine: p53-expressing Ad vector

H101: replication-selective oncolytic Ad

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Time (min)0 5 10 15 20 25 30

Ad

in

blo

od

(%

in

itia

l)

0.01

0.1

1

10

100

Ad clearance from the circulation

Our focus is on systemic gene therapy with adenovirus vectors in vivo

We study

Non-replicating adenovirus vectors (Ad5)

Administered intravenously to rodents

The potential

Gene delivery to any organ or tumor

The reality

Poor pharmacokinetics

Acute toxicity due to innate immune activation

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Research topics

Adenovirus biodistributionOpsonization by plasma proteins

Transduction of hepatocytes

Clearance of vector by Kupffer cells

Safety of adenovirus gene therapyComplement activation

Shock

MAP kinases

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Plasma proteins that opsonize Ad

Adenovirus

IgM

ComplementC1q

ComplementC3b

CoagulationFactor X

Drawn to scale

Ad and the innate immune system

Overall goals:

Understand how innate defense mechanisms affect safety and efficacy

Use this information to develop better vectors 33

Questions?Carolyn Wilson

Porcine endogenous retroviruses

Suzanne Epstein and Graeme PriceUniversal influenza vaccines

Cheng-Hong WeiImmunosuppression by Tregs and MSCsAutoimmunity / tolerance in diabetes

Jakob Reiser and Wu OuLentiviral vector safety and targeting

Andrew ByrnesSafety and efficacy of adenovirus vectors

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