# Presentation: 754264

## Structural Characterization of Gene Transfer Vectors

- BRMAC
- November 16, 2000

**Notes:**
Goal of talk:
1.Provide a framework for this morning’s discussion of structural characterization of gene transfer vectors
2. Presentations that will be given by our invited speakers
3.  Explain the rationale for new FDA proposals regarding characterization of vectors

## Slide 2

**Notes:**
Active INDs
Complex products produced by biological systems
Briefing package:methods used to construct and then produce large amounts of these vectors
Vectors range in size from 3 kb up to 300 kb 
Major FDA concern: structure of the vector, also intermediates used  for such vector construction: examples are  plasmids for adenovirus and  retrovirus.   
Three major points for discussion later today will be 
How much structural characterization should be done
What kinds of structural characterization should be done
When in the course of product development it should be done.  

Two major milestones during product development where this type of analysis is done
before initiation of a clinical trial
before licensure of a product

## Structural Characterization

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

**Notes:**
What is the purpose of structural characterization?

Restriction mapping and PCR are fast, reliable, but low resolution.  
Sequencing gives complete knowledge of the structure but is limited to analysis of one molecular clone or prevalent molecules in a mixture 

## Impact: Safety, Efficacy Production

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

**Notes:**
Replication competent viruses can be detected by assays that detect infectious recombinants

Other types of genetic alterations would not be detected by infectivity assays
Altered transgene:
 change in transgene function, pathogenic
 immunity/autoimmunity 
stop codons
Altered vector backbone:
	 inserted open reading frame
changed transcriptional control

## Sources of Genetic Alterations

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

**Notes:**
In looking at the data we’ve received from sponsors of clinical trials
Two sources of unexpected sequences in gene transfer vectors

I will give examples of each of these types of information
We will have presentations on these topics  

## Incompletely Characterized Intermediates

**ITR**

**ITR**

**AD 5 deleted E3**

**Promoter**
**Transgene**

**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 ?**

**Notes:**
This slide illustrates a problem with insufficient characterization of intermediates used to construct a gene transfer vector.  
Later this morning there will be a presentation by Dr. Zumstein so I will only make a few comments to illustrate  the problem.
Basically, the intermediate plasmid that has been widely used to construct E3 deleted adenoviruses did not contain a simple deletion of E3 sequences.  Instead, the region was replaced by a complex and interesting piece of DNA, mainly from salmon, but also containing nucleotide homologies with human and drosophila DNA.  The insertion also introduces an open reading frame that expressed RNA at a low level.  The ORF has no homologies to known proteins so it could potentially introduce a totally unexpected function along with the intended function of the vector.  

If one only sequenced such a vector but did no further analysis, you would not be able to anticipate problems that might be caused by this extraneous genetic information.

This illustrates importance of sequence analysis of the intermediates used to construct vectors.  
Know what you are starting with.

## Genetic Instability During Manufacture

- Examples
- Generation of replication competent viruses:  retrovirus, adenovirus
- Other mutations

**Notes:**
Generation of replication competent viruses is an important consideration.  I’ll say more about this in a moment.
We have assays in place to detect replication competent recombinants of adenovirus and retrovirus and are 
Always encouraging improvement in these assays.  

## Genetic  Changes in Vector Manufacture

**Genetic  Changes in Vector Manufacture**

**Input**
**Virus**** **

**Notes:**
Mutation, recombination is a stochastic process
Early occurrence: lots of mutated molecules, easy to detect
Late occurrence is more difficult to detect.
We will have presentations from Drs Chamberlin and Levy of this type of phenomena later this morning.
What is signifigance of low level mutated vector?

A surprise that has come from analysis of our March 6 letter to Gene Transfer Sponsors is that as many as 40% of adenovirus vector lots rejected due to RCA. The RCA assays can detect one infectious recombinant  per 10ex9 particles or one part in a billion.  Sequencing analysis  for recombinant vectors has nowhere near this sensitivity. Although we eliminate these vector lots with assays for infectious particles, it makes you realize that there might be a significant amount of other mutations occurring. PCR might be able to detect this level of recombination if you know what to look for.
Even at a sensitivity of one part per billion, when you are giving patients 10e13 particles, you could be giving them 10e10 mutated vector molecules.  This is why we are always pushing for better assays that will lead to improved product characterization.

## 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
-     _<_ 40 KB
- sequence intermediates introduced and flanking regions for vectors > 40 KB
- complete sequence before phase II

**Notes:**

Information we have received concerning incomplete information on vectors and instability during production raises concerns about safety of gene transfer vectors
 We therefore seek to improve the characterization of gene transfer vectors by a variety of methods.  Structural characterization by sequencing analysis is certainly one of the best ways to reach this goal.   We will be asking the committee for discussion of how to best achieve this goal later this morning.
This slide shows our previous recommendation generated in consideration of sequencing technology as it was several years ago.
Tremendous advances in sequencing technology:
Speed has increased
Cost has decreased
These are recommendations we are considering now.  Note that intermediates are also of concern.  For retrovirus, the final product is an RNA virus, presenting technical challenges so compete analysis of the intermediate plasmids is very important.

40 kb cutoff is to include adenovirus

## 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

**Notes:**

Sequence intermediates
sequence vector all or parts depending on size
This would be on Master Virus Bank of Master Vector Bank
Should we also 
Characterize Product lots
Sequence plus other methods
Sequence represents small portion of all the vector molecules
Can detect a maximum of 5-10% impurities if clonal
Therefore  utilize additional methods
PCR: 
very sensitive but limited to predicted portions of vector
Restriction maps:  
low ability to detect small insertions/deletions/point mutations 
5-10% if use southern blot analysis and impurities are clonal
Development of novel approaches desirable.

## Sequence Data Analysis

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

**Notes:**
Another question is what to do with sequence information once obtained
Is comparison to the expected sequence enough?
Talks later today may illustrate the surprising pitfalls that may accompany this approach.
More extensive analysis of sequence data could  include
analysis for open reading frames and comparisons of sequence to both nucleotide and protein databases
Publicly available to anyone with internet access.  Many private data bases available as well.

This is an area in which we will seek some feed back from our advisory panel later this morning.

## Unexpected Sequences

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

**Notes:**
If sequence is obviously a major problem, one option start over, redesign vector. 
However, when faced with unexpected sequences, not always obvious what we should ask of our sponsors
We would like some discussion of this problem from our panel this morning.
Options we consider are:
more rigorous characterization of the vector and its intended  function as well as whatever altered functions may be suggested by the unexpected sequences.
	look for RNA, protein from introduced ORF
New preclinical studies
	unexpected toxicities, antibodies
Proceeding with clinical trial, include additional clinical monitoring

## Summary

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

**Notes:**
I’ve summarized our current recommendations
I’ve also presented some discussion of how we’ve seen unexpected genetic sequences arise in vectors from either insufficient characterization at the beginning or due to the inherent properties of vector production systems.  
Presenters will give more in depth examples of this type of occurance 

Given these findings, as well as in light of technological advances, we are considering more rigorous characterization of  gene transfer vectors.  
Our questions to the committee will address this area