Safety Considerations for Gene Editing and Other Gene ... · Safety Considerations for Gene Editing...
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Safety Considerations for Gene Editing and Other Gene Therapy Products: An FDA Perspective Jakob Reiser, Ph.D. Gene Transfer and Immunogenicity Branch Division of Cellular and Gene Therapies Office of Tissues and Advanced Therapies CBER, FDA
Transcript of Safety Considerations for Gene Editing and Other Gene ... · Safety Considerations for Gene Editing...
Safety Considerations for Gene Editing and Other Gene Therapy Products: An FDA Perspective
Jakob Reiser, Ph.D.Gene Transfer and Immunogenicity Branch
Division of Cellular and Gene TherapiesOffice of Tissues and Advanced Therapies
CBER, FDA
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Gene therapy products regulated by OTAT
Bacterial/yeast4%
Plasmids22%
Adeno13%
‐retro20%
Lenti15%
AAV12%
Pox6%
4HSV2%
Other4%
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560 active Gene Therapy INDs(March 9, 2017)
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Clinical applications of lentiviral vectors
www.fda.gov
Ex vivo applications • T cells to treat HIV infection • CAR T cells to treat leukemias and lymphomas• CD34+ hematopoietic progenitor/stem cells to treat
blood disorders metabolic disorders immunodeficiencies
In vivo applications • Ocular diseases• CNS disorders• Immunotherapy applications
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Safety issues with HIV-1-based lentiviral vectors used in clinical trials
www.fda.gov
Potential to form replication-competent lentivirus
Potential for insertional gene activation/inactivation
Potential for off-target transduction in vivo
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Approaches to improve the safety of lentiviral vectors
www.fda.gov
• Reducing the likelihood to form replication-competent lentivirus
• Narrowing the vector’s tissue tropism
• Directing vector integration to genomic “safe harbor sites”
• Modifying the cell substrate for lentiviral vector production
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Approaches to improve the safety of lentiviral vectors
www.fda.gov
• Reducing the likelihood to form replication-competent lentivirus
• Narrowing the vector’s tissue tropism
• Directing vector integration to genomic “safe harbor sites”
• Modifying the cell substrate for lentiviral vector production
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Emergence of partial recombinants
Packaging construct
Vector construct
Partial recombinant
CMVgag
polRRE
tat
rev
polyA
LTRRRE LTR
gag
transgene
LTR LTRtransgenegag
polRRE
A
B
C
Replication-competent lentivirus?
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Assay to assess formation of partial recombinants
Transduction of HEK 293 cells
Blasticidin (BSD) selection
Characterization of resistant cell clones by PCR and DNA sequencing
Packaging construct
Vector construct
Partial recombinant
CMVgag
polRRE
tat
rev
polyA
BSD
LTRRRE LTR
gag
EGFP
LTR LTREGFPgag
pol
RREBSD
A
B
C ORI
ORI
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Reducing sequence overlaps
www.fda.gov Kuate et al. Human Gene Therapy Methods 25, 126-135 (2014)
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Conclusions• Partial recombinants are rare
• The frequency of partial recombinants is dependent on vector design
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Approaches to improve the safety of lentiviral vectors
www.fda.gov
• Reducing the likelihood to form replication-competent lentivirus
• Narrowing the vector’s tissue tropism
• Directing vector integration to genomic “safe harbor sites”
• Modifying the cell substrate for lentiviral vector production
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Strategy to target lentiviral vector transduction
www.fda.gov
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Strategy to target IL-13R2-positive cells using lentiviral vectors pseudotyped with measles virus-derived hemagglutinin (H) and fusion (F) proteins
Measles virus F protein
Receptor-blind measles virus H protein fused to IL-13
IL-13R2
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Targeting human osteosarcoma cells that conditionally overexpress IL-13R2
www.fda.gov
HOS Uninduced InducedHOS-IL-13R2 HOS-IL-13R2
EGFP
SSC
B
0.2% 0.5% 18.0%
HOS-IL-13R2 HOS
IL-13R2
Cou
nts
uninduced induced
A
Ou et al. Human Gene Therapy Methods 23, 137-147 (2012)
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Large-scale manufacturing of lentiviral vectors pseudotyped with
measles virus H and F proteins
www.fda.gov
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Large-scale manufacturing of lentiviral vectors pseudotyped with
measles virus H and F proteins
www.fda.gov
Samples Transducing units, TUs (%) TUs/mg protein
Input vector samples 100 2.43 × 104 ± 1.21 × 104
Vector eluate 64.36 ± 11.43 2.87 × 106 ± 0.15 × 106
Samples after desalting using Amicon units
60.45 ± 27.64 2.85 × 106 ± 0.33 × 106
100-fold purification
Mustang Q anion exchange membrane chromatography
ÄKTA pure chromatography system
Marino et al. Gene Therapy 22, 64-69 (2015)
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Depiction of a lentiviral vector targeting system involving RNA aptamers
Advantages of aptamer approach:
• RNA sequence space is large
• High affinity aptamer variants are available/can be obtained
• Ease of design/selection/production
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Binding of boxB RNA to lentiviral vectors displaying an λN2-H fusion protein
Bacteriophage
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Binding of boxB RNA to lentiviral vectors displaying an λN2-H fusion protein
A
B
A431 cells
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Transduction of human epidermoid carcinoma A431 cells using lentiviral vector particles displaying an
EGFR-specific RNA aptamer
SSC
EGFP
H‐N + + + +Scaffold + ‐ + +Aptamer + ‐ + +RNase ‐ ‐ + ‐EGF ‐ ‐ ‐ +
2.6% 0.5% 0.5% 0.9%
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Conclusions• Strategy involving measles virus H and F proteins
allowed selective transduction of IL-13R2 positive cells
• Aptamer strategy looks promising but needs optimization
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Approaches to improve the safety of lentiviral vectors
www.fda.gov
• Reducing the likelihood to form replication-competent lentivirus
• Narrowing the vector’s tissue tropism
• Directing vector integration to genomic “safe harbor sites”
• Modifying the cell substrate for lentiviral vector production
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Nuclease-mediated integration of integrase-defective lentiviral vectors (IDLVs) at the
AAVS1 “safe harbor” site on chromosome 19
1 2 3
PuromycinSA pA
1 2 3
AAVS1 locus
Puromycin cassette integrated at AAVS1 site
Left arm Right arm IDLV containing agene of interest
Exon 1 Exon 2 3Intron 1
IDLV encoding an engineered nuclease
PuromycinSA pA
IDLV donor vector
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Double nick strategy involving the AAV2 Rep protein to promote “homologous recombination”
at the AAVS1 locus
1 2 3
Transgene
AAVS1 locus
Left arm Right arm
Exon 1 Exon 2 3
1 2 3Transgene
Rep 78
Nick
Rep 78
Nick
Gonçalves MA et al. Nucleic Acids Res. 40, 3443-3455, 2012
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Rep-mediated integration of IDLVs at the AAVS1 “safe harbor” site
1 2 3
PuromycinSA pA
1 2 3
AAVS1 locus
Puromycin cassette integrated at AAVS1 site
Left arm Right arm IDLV containing apromoterless puromycin cassette
Exon 1 Exon 2 3Intron 1
IDLV encoding Rep 78
p5IEE
PuromycinSA pAp5IEE
IDLV donor vector
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Rep and ZFN-mediated insertion of transgene sequences at the
AAVS1 site mediated by IDLVs
1 2 3PuromycinSA pA
Primers used:
AAVS1-Puro
Puro-Puro
Samples Number of puromycin-resistant clones*
PCR positive clones
Donor 66 ± 12 0% (0/10)
Donor + Rep 275 ± 14 84% (21/25)
Donor + ZFN 200 ± 10 100% (19/19)
p5IEE
*105 cells used for transduction
www.fda.gov
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Conclusions• The overall efficiencies of the nuclease/nickase
approaches need to be improved
• ZFNs and Rep can be toxic if expressed at high levels
www.fda.gov
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Approaches to improve the safety of lentiviral vectors
www.fda.gov
• Reducing the likelihood to form replication-competent lentivirus
• Narrowing the vector’s tissue tropism
• Directing vector integration to genomic “safe harbor sites”
• Modifying the cell substrate for lentiviral vector production
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Design of a HEK 293T cell line bearing a genomic deletion of the SV40 T antigen
coding region
www.fda.gov Heinzel et al. J. Virol. 62, 3738-3746 (1988)
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PCR screensTotal # of clones screened by PCR 176
# of clones negative both for SV40 T‐ag and Km/Neo resistance gene sequences
55
# of clones negative for Km/Neo sequence
37
# of clones negative PCR result for SV40 T‐ag sequence
3Clone #62,Clone #109, Clone #126
# of wild type clones 81
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0.00E+00
5.00E+06
1.00E+07
1.50E+07
2.00E+07
2.50E+07
3.00E+07
Exp1 (TU/mL) Exp2 (TU/mL) Exp3 (TU/mL) Exp4 (TU/mL)
Lentiviral vector production using modified HEK 293T cell clones
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Conclusions• Three out of the 176 cell clones screened
revealed deletions of the T antigen sequence
• Vector titers for the three T antigen knock out clones were lower than those obtained using HEK 293T cells but higher than those obtained using HEK 293 cells
www.fda.gov
AcknowledgmentsTakele ArgawMichael MarinoWu OuBrian IaffaldanoAnna KwilasDahae Hailey BaePingjuan LiMichael MorrealeMartin PanigajSeraphin KuateNahid Parvin
Jizhong Zou, NIH/NHLBIBruce A. Shapiro, NIH/NCIKirill Afonin NIH/NCI Thomas Gaj, ScrippsCarlos Barbas, Scripps (1964-2014)
Funding:Medical Countermeasures Initiative (MCMi) GrantModernizing Science (ModSci) GrantOffice of the Chief Scientist (OCS) Challenge Grant
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Questions?
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