# Presentation: 448929

## Lentiviral Vectors:  Safety Issues

- Daniel Takefman, Ph.D.
- 	Division of Cellular and Gene Therapies
- CBER, FDA

## Retroviridae

- Gammaretrovirus

- env

- Lentivirus

## Gammaretrovirus

- _Lentivirus_

-  Integration into host chromosome

-  No viral genes expressed in
-    target cells

-  Transduces
-   non-dividing cells

-  Efficient adaptation
-   to SIN technology

- Inefficient adaptation
-  to SIN technology

- Potential for recombination resulting
-  in replicating virus with possible pathogenicity

- Can’t transduce
- non-dividing cells

## Lentiviral Vector SystemsUnder Development

- Primate
- Human immunodeficiency virus (HIV)
- Simian immunodeficiency virus (SIV)
- Non-primate
- Feline immunodeficiency virus (FIV)
- Equine infectious anemia virus (EIAV)

## Safety Concerns Specific to Lentiviral Vectors

- Recombination during manufacture may generate a replication-competent lentivirus (RCL)
- - HIV a known human pathogen
- - vesicular stomatitis virus (VSV G) envelope broadens tropism
- Recombination with wild type virus in HIV+ subjects
- Mobilization of lentiviral vector by wild type virus

## Retroviral Recombination: Lessons Learned From Gammaretroviruses

- Homologous recombination can occur when two different RNA’s are packaged into one virion
- Result of reverse transcriptase (RT) template switching (strand transfer)
- Temin, H., et al., PNAS 90(15):6900-3
- Same mechanism shown to occur with HIV RT *in vitro* as well
- Wu, W., et al., J Biol Chem 270(1):325-32

## Retroviral Recombination: lessons 

- Immune suppressed Rhesus monkeys exposed to bone marrow cells transduced with a preparation of RCR positive retroviral vector
- 3/10 developed lymphomas, died within 200 days
- Donahue, R.E., et al., J. Exp. Med. **176**: p. 1125-1135.
- Monkeys had sequences identified as recombinants between vector and helper or between vector and endogenous sequences.
- Vanin, E.F., et al., J. Virology.  **68**(7): p. 4241-4250.
- Purcell, D.F.J., et al., J. Virology.   **70**(2): p. 887-897.

- Retroviral Recombination: lessons
- learned from Gammaretroviral vectors

## Retroviral Recombination: lessons learned from Gammaretroviruses

- Non-homologous recombination occurs at a rate approximately 100-1000-fold lower than homologous recombination
- Reduction in homology between vector and helper sequences will lower likelihood of recombination
- as little as 10 base pairs of nucleotide identity between packaging and vector sequences were sufficient to allow for RCR generation
- Otto, E., et al., Hum Gene Ther 5(5):567-75

## Retroviral Recombination: lessons learned from Gammaretroviruses

- Splitting helper sequences into more than one plasmid (*i.e.*, separation of *env* and *gag-pol*) is likely to decrease the incidence of RCR generation

## Vector Mobilization

- An additional concern with the use of lentiviral vectors in HIV-positive subjects
- Occurs when vector genome is packaged by a wild-type HIV-1 present in the same cell
- Same mechanisms that allow helper sequences to package vector genomes

## Vector Mobilization

- Advantage
- mobilization of a vector designed to inhibit or prevent HIV replication or pathogenesis has been argued to enhance the therapeutic effect
- Disadvantage
- vector spread beyond the intended target tissue may have safety consequences
- co-packaging of wt-type HIV RNA and vector RNA may result in recombination

## How to Address Safety Concerns With Lentiviral Vectors

- Vector design
- 	- incorporate features to decrease likelihood of recombination and mobilization
- 2.	Safety testing during manufacture
- 3.	Preclinical safety studies
- 4.	Clinical monitoring

## 1st Generation Lentivirus Vectors

- Transient transfection of three plasmids in 293T :
- _Packaging plasmid:_
- 	all HIV viral genes, except *env*
- _Envelope plasmid_:
- 	G envelope glycoprotein of vesicular stomatitis virus (VSV G)
- _HIV transfer vector:_
- 	gene or cDNA of interest and the minimal *cis*-acting elements of HIV

## 1st Generation Vectors

- Limited homology between vector and helper sequences
- Separation of helper plasmids
- Still retains HIV accessory genes in the packaging plasmid

## 2nd Generation Vectors

- Elimination of accessory genes from packaging plasmid
- No effect on vector titer
- Retains property of  transduction of many dividing and non-dividing cells
- Increased safety margin

## 3rd Generation Vectors

- Self-inactivating (SIN) vectors
- Deletion in the enhancer region of the 3’ U3 of the long terminal repeat (LTR)
- Results in a transcriptionally inactive vector that can not be converted into a full length RNA
- Reduces likelihood of RCL regeneration
- Hampers mobilization by wild-type HIV
- May reduce risk of tumorigenesis via promoter insertion

## Other Vector Developments

- Splitting the helper sequences onto three separate plasmids by:
- Expressing *rev* on a separate plasmid
- Separation of *gag-pol* coding region onto two plasmids
- Development of stable packaging cell lines based on 3rd generation technology
- Non-HIV vectors: EIAV, SIV, FIV

## How to Address Safety Concerns With Lentiviral Vectors

- Vector Design
- 	- incorporate features to decrease recombination and mobilization
- 2.	Safety Testing during manufacture
- 3.	Preclinical safety studies
- 4.	Clinical Monitoring

## Detection of Replication Competent Lentivirus (RCL)

- Detection of RCL by infectivity assay
- Several passages on permissive cell line
- Endpoint assay for viral sequence (p24 or RT) or transgene sequence
- Positive control?

## Detection of Helper Sequences

- Functional assay
- Tat transfer
- Tat-transactivation of an LTR-reporter gene construct
-  Test for recombination intermediates
- To be discussed this afternoon by Dr. Kappes
- Mol Ther 1(2):47-55

## Detection of Helper Sequences

- Detection of helper sequences in a vector product lot or transduced cells by PCR assay
- Can be very sensitive, not the most biologically relevant assay
- Useful* *for *VSV G* detection

## How to Address Safety Concerns With Lentiviral Vectors

- Vector Design
- 	- incorporate features to decrease recombination and mobilization
- 2.	Safety Testing during manufacture
- 3.	Preclinical Safety Studies
- 4.	Clinical Monitoring

## Use of animal models to assess safety

- Studies to assess mobilization and
- recombination with wild type HIV are difficult.
- Appropriate animal model?
- HIV replicates, but is non-pathogenic in Chimpanzees
- Macaque model appropriate for SIV vector
- Murine model limited due to blocks in HIV replication
- SCID mouse models can serve as “in vivo test tube”, but replication still limited to human cells

## Clinical Monitoring

- Assay for RCL
- How best to do this in HIV+ subject?
- Assay for recombination with wild type HIV
- Assay for changes in patient wt HIV

## Lentiviral Safety Concerns: Conclusions

- Recombination during manufacture
- Vector design
- Safety testing
- Recombination with wild type virus in HIV+ subjects
- Clinical monitoring

## Lentiviral Safety Concerns:	Conclusions

- Mobilization by wild type virus in HIV+ subjects
- In vitro assay
- Preclinical animal model
- Clinical monitoring