Date post: | 14-Dec-2015 |
Category: |
Documents |
Upload: | jermaine-dutton |
View: | 220 times |
Download: | 4 times |
Therapeutics, Inc.
INTROGENINTROGEN
DNA Sequencing of Viral Gene Therapy Vectors During Pre-Clinical and Clinical
Development
INTROGENINTROGEN
Forward Looking Statement
During the course of this presentation, I may make
projections or other forward-looking statements regarding
future events. I wish to caution you that such statements
are just predictions and that actual events or results may
differ materially. Further information on factors that could
affect Introgen’s results can be found in the Company’s
filings with the Securities and Exchange Commission,
including Introgen’s registration statement on Form S-1.
INTROGENINTROGEN
Vector Sequences: Dichotomy Between Belief and Reality
• Barring sequencing data, the predicted origins and sequence of a vector or plasmid should be considered to be tentative.
•History of sequencing for RPR/INGN 201 (Ad5CMV-p53 )
INTROGENINTROGEN
Method for Generating Ad5CMV-p53
ShuttleVector~7 kb
pJM17~40 kb
0
pBR322
Ad-L
Ad-R
Ad-L
Ad-R
CMV-p53 Ad
p53p53
E4
E2 E3(E1 del)
L
INTROGENINTROGEN
Ad5CMV-p53: Starting Reagents
• Ad5CMV-p53 and the reagents used to make it originated from the laboratory of Dr. Jack Roth at MDACC.
• Documentation on the constructs was from academic scientific lab notebooks.
• Shuttle vector - maps with restriction sites.
• pJM17 was derived from the Ad5 dl309 adenovirus.
• Ad5 dl309 was constructed to remove several restriction sites in the Ad5 genome.
• Ad5 dl309 was selected as a viable virus, and grows as well as wild-type Ad5.
INTROGENINTROGEN
Ad5CMV-p53: Starting Reagents (continued)
• At this stage we knew the historic building blocks that went into Ad5CMV-p53, and we knew that there had been changes made to several restriction sites in the Ad5 derived part of the Ad5CMV-p53.
INTROGENINTROGEN
Ad5CMV-p53 Sequence, Version 1
• Rationale - To confirm the sequence of the expression cassette.
• Methods - Manual sequencing of expression cassette.
• Timing - Early in technology transfer from academic laboratory to Introgen’s research laboratories. Prior to initiation of Phase I trials.
INTROGENINTROGEN
Ad5CMV-p53 Sequence, Version 1(continued)
• Conclusions - DNA sequence of expression cassette was as expected.
• New Information - The polylinker sequences in the expression cassette were now defined.
INTROGENINTROGEN
Ad5CMV-p53 Sequence, Version 1(continued)
Ad-L CMV p53 pA Ad-R
p53p53
E4
E2 E3(E1 del)
L
INTROGENINTROGEN
Ad5CMV-p53 Sequence
p53p53
E4
E2 E3
In November 1995 Frank Graham and colleagues published a paper regarding changes in E3 region of dl309.
INTROGENINTROGEN
Ad5CMV-p53 Sequence, Version 2
• Methods -GLP sequencing of expression cassette cloned out of clinical lot of Ad5CMV-p53 , and sequencing of flanking viral DNA sequences directly from Ad5CMV- p53 viral DNA.
• Timing - Summer of ‘96, Phase I ongoing
INTROGENINTROGEN
Ad5CMV-p53 Sequence, Version 2(continued)
•Conclusions - The expression cassette sequence agreed with previous sequence data.
•New information -
– Fully defined sequence of polylinkers between Ad sequences and the expression cassette.
– About 300 bp of the left adenoviral flank of Ad5CMV-p53 has more similarity to Ad2 than to Ad5.
INTROGENINTROGEN
Ad5CMV-p53 Sequence
• In May of ‘98 a paper was published by the group of Estuardo Aguilar-Cordova (Gingras et al) which had a sequence of the E3 insertion/deletion of pJM17 that was slightly different than the sequence of this region of dl309 from the Graham lab.
• Exact sequence of the E3 insertion/deletion event was unclear.
INTROGENINTROGEN
Ad5CMV-p53 Sequence, Version 3
• Method - GLP sequencing of all of Ad5CMV-p53.
• Rationale - Complete sequence desired prior to initiation of pivotal trials.
• Timing - April 1999; late Phase II.
INTROGENINTROGEN
Ad5CMV-p53 Sequence, Version 3(continued)
Conclusions
• p53 expression cassette - No changes.
• E3 region - Sequence agreed with Gingras et al.
• Aside from E3 region, there were 23 discrepancies between the predicted sequence, which was based on published data, and the sequence ascertained in this sequencing of Ad5CMV-p53.
INTROGENINTROGEN
Ad5CMV-p53 Sequence, Version 3(continued)
Discrepancies between published (Genbank) sequences and actual sequence found for Ad5CMV-p53
Non-coding/silent 10
Amino Acid change 9
Change ORF extent 4
INTROGENINTROGEN
Ad5CMV-p53 Sequence, Version 3(continued)
E3 Region, Ad5CMV-p53, predicted ORFs 12.5K 6.7 19K 11.6K fusion novel
6 bp del Insertion/deletion
E3 Region, wild-type Ad5, predicted ORFs
12.5K 6.7 19K 11.6K 10.4K 14.5K 14.7K
INTROGENINTROGEN
Ad5CMV-p53 Sequence, Version 3(continued)
Predicted changes to E3 proteins of Ad5 vs Ad5CMV-p53.
Protein Function Change
6.7K Unknown 6 bp deletion; made at reducedlevels, and only one of thenormal glycosylated forms isfound.
10.4K prevents TNF-mediated cytolysis
deletion of 18 aa’s fromC-terminus, addition of 27amino acids.
14.5K prevents TNF-mediated cytolysis
ORF completely deleted.
14.7K prevents TNF-mediated cytolysis
ORF completely deleted.
Novel Novel ORF, no similarity toknown proteins.
INTROGENINTROGEN
Ad5CMV-p53 Sequence, Version 3(continued)
Nature of sequences inserted into the E3 region :• High degree of similarity between a small part of the inserted sequence and a salmon DNA sequence in Genbank (92% over 135 bp).• From Gingras et al, we know that DNA from this insert hybridizes to salmon DNA and not human DNA.• From studies at Introgen and from Gingras et al we know that RT-PCR detects RNA made from this region.
Insertion/Deletion region of E3 (646 base pairs)
Novel ORF
Homology
INTROGENINTROGEN
Ad5CMV-p53 Sequence
p53p53
E4
E2 E3(E1 del)
L
INTROGENINTROGEN
Conclusions
• Introgen has sequenced all of Ad5CMV-p53, prior to initiating phase III trials.
• Until sequencing is completed, there will be uncertainties about the exact identity of almost all plasmids and vectors.
Therapeutics, Inc.