# Presentation: 225336

## Predicting Lentiviral Vector Safety In Vivo

## Challenges for for clinical testing

-   Tremendous advances in vector safety design while
-     retaining efficient gene transfer in vivo.

- Status of field

## Emergence of RCL is the Principal Safety Concern

- (transduction/primary recombination)

- LTR-gag-pol-env-LTR

- LTR-gag-pol-LTR

- env-

- ?

## Safety Considerations

-   likely
-    - experience with retrovirus vectors
-    - utilized for reverse transcription

-   underpins generation of RCL/safety

## Safety Considerations

-   *in vitro* = generation of LTR-gag-pol-env-LTR-
-      	        like recombinants

-   *in vivo* = failure in vector safety and/or QC

## What Requisite Biosafety Measures

-   QA/QC testing (LV stocks)

- a.  PCR assay

- b.  RCL assay

- c. Gag-Pol recombinant assay

## RCL assay

-   Detects replication competent recombinants

-   Not informative of non-RCL recombinants

- _Advantages:_

- _Disadvantages:_

- ?  Functionality or replication potential of recombinants

- ?  How recombinants will interact with host

- ?  How the host will interact with the recombinants

-   Not predictive against the emergence of RCL in vivo

- ?  Genetic composition of recombinants

- ?  Risk to the treated individual

## PCR assay

-   Detects vector- and/or packaging-specific DNA

-   Specificity

- _Advantages:_

- _Disadvantages:_

-   Biologically non-specific

## Gag-Pol Recombinant Assay

-   Enables monitoring of vector stocks for pre-RCR
-     recombinants - Specifically, recombinants with a
-     functional gag-pol coding region

- _Significance:_

## Gag-Pol Recombinant Assay

-   Shows gag-pol-vector recombinants are produced

-   Functional gag-pol is required for the recombinant to
-     generate RCL in vivo

-   Without functional gag-pol (LTR-gag-pol-LTR), RCL
-     cannot be generated in primary transduced cells

-   Thus, in vitro monitoring for functional gag-pol-containing
-     recombinants provides a tangible way to analyze LV stocks
-     in vitro for their potential to generate RCL in vivo

## QC surrogate

- RCL?

- Recombination

- Recombination in vivo?

- (gag-pol recombinants)

- Hypothesis

## Analysis of Genetic Recombination

-   Genetic Recombination Underpins the generation of RCL

-   Approach:
-   Detect
-   Enrich
-   Characterize
-       - genetically
-       - biologically

## Approach for Analyzing Genetic Recombination

**HeLa-puro**

## Approach for Analyzing Genetic Recombination

- 

**Selection and characterization of**
**recombinant-containing cells**

**tat**

- LTR

- ga

- LTR

- RRE

**puro**

- LTR

**HeLa-puro**

- 

**Recombinant**

- LTR

- ga

- puromycin

## +

- SIN vector

- 3rd generation packaging construct

- Trans-lenti vector

**+**

- State-of-the-Art Vector Components

## Split Function Lentiviral Vector System

- Vector Construct

- Packaging Construct

- Env Construct

**VSV-G**

- CMV

- tat

- SD

- rev

- rev

- tat

- RRE

- poly A

- gag

- CMV

- LTR

- Ga

- RRE

- CMV

**GFP**

- LTR

- 

- poly A

## Lentiviral Vector tat Transfer

**HeLa-Puro**

**Puromycin Selection**

**LTR-puro**

- tat

-   Generation of tat-containing recombinants

## Lentiviral Vector gag Transfer

**HeLa-Puro**

**LTR-puro**

- - Nevirapine

- + Nevirapine

- Mock

- Lentiviral Vector

- 0 CFU

- 0 CFU

- 540 CFU

- Puromycin Selection

- Infection

- 107 IU

**gag-pol orf ?**

-   Generation of recombinants with functional gag-pol genetic structure

- pVSV-G

- ptat/rev

## Genetic Analysis of Recombinant Proviral DNA

## 5’ Sequence Analysis of Genetic Recombinants

- orf (100%)

- gag

- R

- U5

## Recombination within the Poly(A) Tract of the Packaging Construct 

**U3**

**R**

**... ****AAUGAAA... AAAAAAAAAAAAAAAAAAAAAA... **

**(47)**

**(53)**

**(63)**

**Packaging construct**

**r**

**u5**

**u3**

**AAAA**

- (pA signal)

**Vector**

- cDNA

**RNA template**

**n**

- Recombination within the Poly(A) Tract of the Packaging Construct

**mRNA**

## 3’ Sequence Analysis of Genetic Recombinants

- RRE

- U3

- R

- U5

- tat/rev

- Packaging Construct (3’ end)

- Vector (3’ LTR)

- pA signal

**PPT**

** **AAGAGGAGGAGGAGGTGGG...GGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGA

** **AAGAGGAGGAGGAGGTGGG...GGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGA

-  ATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGA

** **AAGAGGAGGAGGAGGTGGG...GGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGA

- (x 6)

- (x 1)

- (x 1)

- (x 1)

- 108

- 32

**U3**

## Trans-Lentiviral Vector System

- Packaging construct

- Trans-enzyme construct

- LTR

- pA

- vpr

- RT

- IN

## TLV Impairs Gag Transfer/DNA mobilization

**HeLa-Puro**

**LTR-puro**

- Puromycin selection

- ?

- 107 IU

- Infection

-   Trans-lenti vector does not generate detectable recombinants

-   Block in DNA mobilization due to trans-RT-IN

-   Absence of functional Gag-Pol (RT-IN) blocks mobilization

- pVSV-G

- ptat/rev

## Summary: Analysis of lentiviral vector recombination

-     gene transfer vector

-   These particles package mRNA and if pseudotyped, mobilize the mRNA
-     to other target cells where it is reverse transcribed and integrated

-    The expression of the integrated gag and pol gene produces progeny
-     env-deficient recombinant lentivirus particles

-   Integrated recombinants express viral proteins including, Tat, Gag,
-     and the entire Gag-Pol precursor polyprotein

## Summary: Analysis of lentiviral vector recombination

-   Recombination within the mRNA poly(A) tract:
-  confirmed  genetic recombination during reverse transcription
-    in the infected cell
-   suggested that removing homologous sequences from the vector and
-    packaging construct may not be sufficient to prevent recombination
-   may represent a mechanism by which genes without homologous
-    sequence can be mobilized, including endogenous genes (Huang et al.,
-    Cell: 44:936, 1986; Raines et al., J. Virol. 62:2437, 1988)

## +

- gag

- CMV

- RRE

- pA

- SIN vector

- 3rd generation packaging construct

- Trans-lenti

**+**

- Tat-Independent Analysis of Genetic Recombination

- PR

- RT

- IN

- pA

- LTR

- RRE

- vpr

- RT

- IN

## Gag-Pol-Dependent DNA Mobilization Assay

**HeLa-tat**

- pVSV-G

- Infection

- 

- Recombinant

- Puromycin selection

- CMV-tat

- 108  - 3rd gen.
- 108  - 3rd gen/SIN
- 109  - trans-lenti

- ptat/rev

## Gag-Pol Dependent DNA Mobilization

- trans-lenti

- SIN + 3rd gen.

- 3rd gen.

## Conclusions

-     recombinants with functional gag-pol capable of mobilizing DNA

-   Since a functional gag-pol genetic structure is absolutely required for
-     the generation of RCL, monitoring vector stocks for the production
-     of env-minus gag-pol-containing recombinants may serve as an in
-     vitro surrogate marker to control against generating RCL in vivo.

-   Separating RT and IN from the packaging construct decreases the
-     frequency of regeneration of a functional gag-pol structure (and DNA
-     mobilization) by at least 2 orders of magnitude

**Conclusions**

-   The trans-lentiviral vector design is particularly amenable for
-     functional gag-pol QC testing

## Utility of in vitro monitoring for functional 

- _Utility_ of in vitro monitoring for functional
- gag-pol-containing recombinants to QC against
- the _potential_ of vector stocks to produce RCL in vivo

- Theoretical ?

- Biologically significant ?

## Gp120-Receptor-Independent Mechanism(s) for HIV-1 Infection

-   Cellular membrane proteins are incorporated into virion during
-     budding (Arthur et al., Science 258:1935, 1992)

-   Interaction between cell-derived membrane protein and receptor
-     on cell surface facilitates initial binding  (Wu et al., submitted)

-   The initial binding of HIV to target cells does not require Env-
-     receptor interaction  (Mandor et al., J. Virol. 72:3623, 1998;
-     Wu et al., submitted)

-   HIV Env-independent infection of CD4-minus epithelial cells
-     (Duan et al., J. Virol. 74:10994, 2001)

-   Interaction between cell-derived membrane protein and a cellular
-     receptor can support HIV-1 infection  (Enders et al., Science 278:1462,
-     1997; Mebatsion et al., cell 90:841, 1997; Schnell et al., Cell 90:849, 1997)

## Perpetuate Risk for RCL

- Infection

- Env-minus
- virions

-   Each cycle of replication represents an additional opportunity
-     for genetic recombination and the generation of RCL

## Analysis of Env-Minus Vector Infectivity 

- Env-minus
- vector

- CD4-minus

- 2.  cDNA synthesis

- 4.  Infection/proviral formation

- 3.  Entry route

- Analysis of Env-Minus Vector Infectivity

- 1.  Virion binding

## Attachment Independent of CD4 & gp120

- HeLa CD4+

- HeLa CD4-

- Env+

- Env-

## Vector DNA Synthesis Independent of gp120-CD4

- Receptor-Mediated Entry

- R-U5

- R-gag

- Infection:

- 3TC:

- 293

- HeLa

- JC53

- -

- -

- -

- -

- +

- -

- -

- +

- -

- -

- +

- -

- +

- +

- +

- +

- +

- +

## Vector DNA Synthesis in Acidified Endosomes

## Analysis of Vector Infectivity in CD4-minus Cells 

- Vector
- particles

- HT-1080

- Tu139

- BFLA1+

- BFLA1—

- BFLA1+

- BFLA1—

- HeLa

- Env

- Env

- VSV-G

- 7.3x103

- 1.8x104

- 2.4x106

- 4.8x102

- 2.5x103

- 7.7x104

- 2.8x103

- 3.5x103

- 3.3x103

- 5.0x103

- 1.5x105

- JC53

- ND

- ND

- 0

- 0

- ND

- ND

- ND

## Promise of Lentiviral Vectors for Gene Therapy 

- Central Nervous System Disorders

- Hematopoietic Stem Cells

- Eye Diseases

*Proc. Natl. Acad. Sci.* 94:10319, 1997

## Slide 40

## Transduction of Neurons In Vivo

- Transduction of Neurons *In Vivo*

## Transduction of  Retinal Pigment Epithelium

## Conclusions

-   In vitro monitoring for functional gag-pol-containing recombinants
-     may serve as a surrogate marker to control against the emergence
-     of RCL in vivo

-   The trans-lentiviral vector design splits the gag-pol function  and
-     therefore, is particularly well suited for gag-pol QC monitoring

-   The formation of proviral DNA recombinants with a functional
-     gag-pol coding region may increase the risk for RCL

## In vitro Monitoring to Predict the Potential for

- Generating RCL in vivo

- RCL?

- Recombination in vivo?

- QC surrogate

- (gag-pol recombinants)

## Acknowledgments

- John Wakefield

- Hongmei Liu

- Lilin Lai

- Yimin Wang

- Tranzyme Inc.

- UAB

- Jean Bennett

- U. Penn.

- Xiaoyun Wu

- Tim Townes

- WenYong Chen

- Lori McMahon

## Safety Considerations

-   Genetic recombination

-   Unknown pathogenicity or pathogenic potential of
-     recombinant lentiviral vectors (including human and
-     non-human)

-   Quality assurance & quality control (QA/QC)

-   Generation of RCL in vivo

- * The ability of lentiviral vectors to infect non-dividing
-    cells raises safety issues for which we can not drawl
-          upon prior experience with retroviral vectors