Structural Characterization of Gene Transfer Vectors

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Structural Characterization of Gene Transfer
Vectors

• BRMAC
• November 16, 2000

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(No Transcript)
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Structural Characterization

• Purpose
• vector produced has expected characteristics
• vector production is reliable
• Methods
• Restriction map
• PCR
• Nucleotide sequence

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Impact Safety, Efficacy Production

• Safety
• replication competent virus
• altered vector backbone
• altered transgene
• Efficacy
• Production process

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Sources of Genetic Alterations

• Incompletely characterized vector backbones
• intermediates not always well characterized
• Instability during vector manufacture
• recombination/rearrangements
• mutation

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Incompletely Characterized Intermediates
Promoter Transgene
ITR
ITR
AD 5 deleted E3
646 bp insert Interrupts E3B 10.5K, 14.6K, 14.7K
genes Salmon, human, Drosophila homologies
132-aa ORF, no protein homologies RNA detected
by RT-PCR Protein expression ?
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Genetic Instability During Manufacture

• Examples
• Generation of replication competent viruses
  retrovirus, adenovirus
• Other mutations

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Genetic Changes in Vector Manufacture
Input Virus
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Recommendations

• Current
• Early phase of development
• sequence appropriate portions of vectors
• or restriction maps plus protein characterization

• Proposed
• Before phase I
• complete sequence of intermediates and vectors
• lt 40 KB
• sequence intermediates introduced and flanking
  regions for vectors gt 40 KB
• complete sequence before phase II

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Timing and Extent of Characterization

• Before phase I
• sequence intermediates and all or portion of
  vector before initiation of clinical trial
• Product lot
• sequence
• other methods

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Sequence Data Analysis

• Comparison to expected sequence
• Analysis for open reading frames
• Comparisons to nucleotide and protein data bases

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Unexpected Sequences

• What additional experiments should be done to
  examine effects of unexpected sequences?
• Vector characterization
• Preclinical studies
• Patient follow-up

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Summary

• Current recommendations for vector sequencing
• Current information reveals unexpected genetic
  material
• Considering more rigorous structural
  characterization