Predicting Lentiviral Vector Safety In Vivo

1
Predicting Lentiviral Vector Safety In Vivo
2
Status of field

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

Challenges for for clinical testing

• Devise an approach(s) for safe administration
  of vector

3
Emergence of RCL is the Principal Safety Concern
(transduction/primary recombination)
?
env-
LTR-gag-pol-env-LTR
LTR-gag-pol-LTR
4
Safety Considerations
Genetic recombination

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

• underpins generation of RCL/safety

5
Safety Considerations
Generation of RCL in vivo

• in vitro generation of LTR-gag-pol-env-LTR-
• like recombinants

• in vivo failure in vector safety and/or QC

6
What Requisite Biosafety Measures

• QA/QC testing (LV stocks)

a. PCR assay
b. RCL assay
c. Gag-Pol recombinant assay
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RCL assay
Advantages

• Detects replication competent recombinants

Disadvantages

• Not predictive against the emergence of RCL in
  vivo

• Not informative of non-RCL recombinants

? Genetic composition of recombinants
? Functionality or replication potential of
recombinants
? How the host will interact with the
recombinants
? How recombinants will interact with host
? Risk to the treated individual
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PCR assay
Advantages

• Detects vector- and/or packaging-specific DNA

Disadvantages

• Biologically non-specific

• Specificity

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Gag-Pol Recombinant Assay
Advantages

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

Significance
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Gag-Pol Recombinant Assay
Significance

• Shows gag-pol-vector recombinants are produced

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

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

• 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

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Hypothesis
Recombination
QC surrogate
(gag-pol recombinants)
Recombination in vivo?
RCL?
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Analysis of Genetic Recombination

• Genetic Recombination Underpins the generation
  of RCL

• Approach
• Detect
• Enrich
• Characterize
• - genetically
• - biologically

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Approach for Analyzing Genetic Recombination
HeLa-puro
14
Approach for Analyzing Genetic Recombination
HeLa-puro
Recombinant
y
tat
LTR
LTR
ga
y
ga
LTR
RRE
puro
LTR
puromycin
Selection and characterization of recombinant-cont
aining cells
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State-of-the-Art Vector Components
3rd generation packaging construct
SIN vector
Trans-lenti vector

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Split Function Lentiviral Vector System
Packaging Construct
tat
rev
rev
SD
RRE
poly A
CMV
gag
tat
Vector Construct
y
LTR
Ga
RRE
CMV
GFP
LTR
Env Construct
VSV-G
CMV
poly A
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Lentiviral Vector tat Transfer
HeLa-Puro
tat
107 IU
LTR-puro
Puromycin Selection

• Generation of tat-containing recombinants

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Lentiviral Vector gag Transfer
HeLa-Puro
107 IU
gag-pol orf ?
LTR-puro
Infection
Puromycin Selection
pVSV-G
ptat/rev
Mock
Lentiviral Vector
- Nevirapine
Nevirapine
0 CFU
0 CFU
540 CFU

• Generation of recombinants with functional
  gag-pol genetic structure

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Genetic Analysis of Recombinant Proviral DNA
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5 Sequence Analysis of Genetic Recombinants
orf (100)
gag
R
U5
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Recombination within the Poly(A) Tract of the
Packaging Construct
Packaging construct
(47)
(53)
(63)
mRNA
AAUGAAA AAAAAAAAAAAAAAAAAAAAAA...
(pA signal)
r
u5
u3
cDNA
RNA template
AAAA
U3
R
n
Vector
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3 Sequence Analysis of Genetic Recombinants
Packaging Construct (3 end)
Vector (3 LTR)
pA signal
RRE
U3
R
U5
tat/rev
108
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U3
PPT
(x 6)
AAGAGGAGGAGGAGGTGGG...GGCAGCTGTAGATCTTAGCCACTTTTT
AAAAGAAAAGGGGGGACTGGA
(x 1)
AAGAGGAGGAGGAGGTGGG...GGCAGCTGTAGATCTTAGCCACTTTTT
AAAAGAAAAGGGGGGACTGGA
(x 1)
AAGAGGAGGAGGAGGTGGG...GGCAGCTGTAGATCTTAGCCACTTTTT
AAAAGAAAAGGGGGGACTGGA
(x 1)
ATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGA
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Trans-Lentiviral Vector System
Packaging construct
Trans-enzyme construct
vpr
pA
LTR
RT
IN
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TLV Impairs Gag Transfer/DNA mobilization
107 IU
HeLa-Puro
LTR-puro
?
Infection
Puromycin selection
pVSV-G
ptat/rev

• 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

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Summary Analysis of lentiviral vector
recombination

• Recombination occurs between the lentiviral
  packaging construct and
• gene transfer vector

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

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

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

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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 44936, 1986 Raines et al., J. Virol.
  622437, 1988)

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Tat-Independent Analysis of Genetic Recombination
3rd generation packaging construct
PR
RT
IN
RRE
pA
CMV
gag
SIN vector
Trans-lenti

vpr
RT
IN
pA
LTR
RRE
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Gag-Pol-Dependent DNA Mobilization Assay
108 - 3rd gen. 108 - 3rd gen/SIN 109 -
trans-lenti
HeLa-tat
CMV-tat
Recombinant
Infection
y
pVSV-G
Puromycin selection
ptat/rev
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Gag-Pol Dependent DNA Mobilization
trans-lenti
SIN 3rd gen.
3rd gen.
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Conclusions

• The 3rd generation packaging construct and SIN
  vector generate
• recombinants with functional gag-pol capable
  of mobilizing DNA

• 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

• 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.

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

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

• Cellular membrane proteins are incorporated
  into virion during
• budding (Arthur et al., Science 2581935,
  1992)

• The initial binding of HIV to target cells does
  not require Env-
• receptor interaction (Mandor et al., J.
  Virol. 723623, 1998
• Wu et al., submitted)

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

• Interaction between cell-derived membrane
  protein and a cellular
• receptor can support HIV-1 infection (Enders
  et al., Science 2781462,
• 1997 Mebatsion et al., cell 90841, 1997
  Schnell et al., Cell 90849, 1997)

• HIV Env-independent infection of CD4-minus
  epithelial cells
• (Duan et al., J. Virol. 7410994, 2001)

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Perpetuate Risk for RCL
Env-minus virions
Infection

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

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Analysis of Env-Minus Vector Infectivity
Env-minus vector
CD4-minus
3. Entry route
1. Virion binding
4. Infection/proviral formation
2. cDNA synthesis
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Attachment Independent of CD4 gp120
HeLa CD4
HeLa CD4-
Env
Env-
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Vector DNA Synthesis Independent of
gp120-CD4 Receptor-Mediated Entry
293
HeLa
JC53
-
-
-
-
-
-



3TC
-
-
-






Infection
R-U5
R-gag
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Vector DNA Synthesis in Acidified Endosomes
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Analysis of Vector Infectivity in CD4-minus Cells
HT-1080
Tu139
Vector particles
HeLa
JC53
BFLA1
BFLA1
BFLA1
BFLA1
DEnv
7.3x103
4.8x102
2.8x103
3.3x103
0
0
Env
1.8x104
2.5x103
3.5x103
5.0x103
1.5x105
ND
VSV-G
2.4x106
7.7x104
ND
ND
ND
ND
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Promise of Lentiviral Vectors for Gene Therapy
Hematopoietic Stem Cells
Central Nervous System Disorders
Eye Diseases
Proc. Natl. Acad. Sci. 9410319, 1997
40
(No Transcript)
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Transduction of Neurons In Vivo
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Transduction of Retinal Pigment Epithelium
(Histologic Assessment)
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Conclusions

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

• 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

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In vitro Monitoring to Predict the Potential
for Generating RCL in vivo
QC surrogate
(gag-pol recombinants)
Recombination in vivo?
RCL?
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Acknowledgments
Tranzyme Inc.
UAB
U. Penn.
John Wakefield
Jean Bennett
Lilin Lai
Hongmei Liu
Yimin Wang
Xiaoyun Wu
Tim Townes
WenYong Chen
Lori McMahon
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Safety Considerations

• Insertional mutagenesis

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

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

• Genetic recombination

• Generation of RCL in vivo

• Quality assurance quality control (QA/QC)