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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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