Gene Therapy for EB: State of the Art

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Gene Therapy for EBState of the Art

• Anna L. Bruckner, MD
• Stanford University School of Medicine

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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?

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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

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Gene transfer technology

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

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Khavari PA et al. J Intern Med 20022521-10.
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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

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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

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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.

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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.

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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.

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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

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Ortiz-Urda S et al. Nat Med 200281166-70
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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.

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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.

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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

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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

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Protein therapy, cont.

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

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Woodley DT et al. Nat Med 200410693-5.
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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?

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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.

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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.