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Gene Therapy for EB: State of the Art

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2/11 patients developed leukemia-like syndrome. Hacein-Bey-Abina S et al. 2003. ... Inserts DNA containing an attB sequence into genomes containing attP sites ... – PowerPoint PPT presentation

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Title: Gene Therapy for EB: State of the Art


1
Gene Therapy for EBState of the Art
  • Anna L. Bruckner, MD
  • Stanford University School of Medicine

2
Overview
  • Why are recessive forms of EB ideal candidates
    for gene transfer or protein therapy?
  • What is gene transfer technology, and how does it
    work?
  • What has been done so far?
  • What looks promising for the future?

3
JEB and RDEB
  • Debilitating current treatments do not modify
    disease course
  • Mutations in a single gene lead to absent or
    nonfunctional proteins
  • Replacing the defective gene (or protein) will
    repair the problem

4
Gene transfer technology
  • The insertion of a normal gene into a somatic
    cell in order to replace a missing or abnormal
    gene product

5
Khavari PA et al. J Intern Med 20022521-10.
6
Pros and Cons
  • In vivo
  • Simple and direct
  • Low efficiency of gene transfer
  • Effects short-lived
  • Safety concerns
  • Ex vivo
  • Cumbersome
  • Expensive
  • Potentially painful and scarring
  • Higher efficiency of gene transfer confirmed in
    culture
  • Control safety

7
Viral vector targeting keratinocytes
  • Modified retrovirus used to insert gene into
    target cell
  • Up to 8 kb genetic material can be transferred
  • Efficient integration into host DNA
  • Rarely immunogenic

8
Retroviral vector
  • Approach successful for JEB
  • LAMB-3
  • Dellambra E et al. 1998.
  • Robbins PB et al. 2001.
  • COL17A1
  • Seitz CS et al. 1999.
  • ITGB-4
  • Dellambra E et al. 2001.

9
Retroviral vector pitfalls
  • Limitation on size of transferable genes
  • Gene expression not sustained
  • Potential for insertional mutagenesis
  • Clinical trial for X-linked SCID
  • 2/11 patients developed leukemia-like syndrome
  • Hacein-Bey-Abina S et al. 2003.

10
JEB nonviral gene transfer
  • LAMB3 gene has been successfully transferred to
    JEB keratinocytes using nonviral methods and
    corrects JEB in vivo
  • øC31 bacteriophage integrase
  • Ortiz-Urda S et al. Hum Gene Ther 200314923-8.
  • Sleeping Beauty transposable element
  • Ortiz-Urda S et al. Gene Ther 2003101099-1104.

11
RDEB øC31 bacteriophage integrase
  • Inserts DNA containing an attB sequence into
    genomes containing attP sites (pseudo-attP sites
    in mammals)
  • Can insert DNA gt 10 kb in size
  • COL7A1 transferred into RDEB keratinocytes
  • Transfected keratinocytes expressed type VII
    collagen
  • Type VII collagen restored BMZ structure and
    functional
  • Results lasted at least 14 weeks in vivo

12
Ortiz-Urda S et al. Nat Med 200281166-70
13
RDEB Lentiviral vector transfer
  • Slightly modified type VII collagen cDNA (8.85
    kb) transferred via lentivirus to RDEB
    keratinocytes and fibroblasts
  • Type VII collagen expression restored
  • BMZ structure and function restored
  • Chen et al. Nat Genet 200232670-5.

14
RDEB Fibroblasts
  • RDEB-fibroblasts engineered to overexpress type
    VII collagen restore type VII collagen at the DEJ
  • Ortiz-Urda S et al. J Clin Invest 2003111251-5.
  • Normal and gene-corrected fibroblasts restore
    type VII collagen at the DEJ
  • Woodley DT et al. J Invest Dermatol
    20031211021-8.

15
Keratinocytes vs. fibroblasts
  • Keratinocytes
  • Fragile
  • Use immediately
  • Wounding needed for engraftment
  • Specialized wound care post-op
  • Risk for scarring
  • Fibroblasts
  • Robust
  • Can be frozen
  • Delivery via intradermal injection

16
Protein therapy for RDEB
  • Type VII collagen purified from culture medium of
    gene-corrected RDEB fibroblasts
  • Injected intradermally
  • Stably incorporated into BMZ of athymic mice for
    at least 3 months and immunocompetent mice for at
    least 6 weeks
  • 6/10 developed antibodies to type VII collagen
    but were asymptomatic

17
Protein therapy, cont.
  • BMZ of RDEB skin grafted onto mice also restored,
    blistering stopped
  • Results lasted at least 2 months following a
    single injection

18
Woodley DT et al. Nat Med 200410693-5.
19
Unanswered questions
  • Gene transfer vs. protein therapy? Or cultured
    normal fibroblasts?
  • What is the potential for developing a clinically
    significant immune response to type VII collagen?
  • How will giving back type VII collagen affect the
    risk for developing SCC?

20
References
  • Reviews
  • Hengge UR. Clin Dermatol 200523107-14.
  • Khavari PA et al. J Int Med 20022521-10.
  • Ferrari S et al. Clin Dermatol 200523430-6.
  • JEB
  • Dellambra et al. Hum Gene Ther 199891359-70.
  • Dellambra et al. J Biol Chem 200127641336-42.
  • Robbins PB et al. Proc Natl Acad Sci U S A
    2001985193-8.
  • Seitz CS et al. Gene Ther 1999642-7.
  • Ortiz-Urda S et al. Hum Gene Ther 200314923-8.
  • Ortiz-Urda S et al. Gene Ther 2003101099-1104.
  • SCID
  • Hacein-Bey-Abina S et al. Science 2003302415-9.

21
References, cont.
  • RDEB
  • Ortiz-Urda S et al. Nat Med 200281166-70.
  • Ortiz-Urda S et al. J Clin Invest 2003111251-5.
  • Chen et al. Nat Genet 200232670-5.
  • Woodley DT et al. J Invest Dermatol
    20031211021-8.
  • Protein therapy
  • Woodley DT et al. Nat Med 200410693-5.
  • RDEB and SCC
  • Ortiz-Urda S et al. Science 20053071773-6.
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