88 site specific genome modification of human primary t lymphocytes to improve the safety and efficacy of adoptive immunotherapy

88 Site Specific Genome Modification of Human Primary T Lymphocytes To Improve the Safety and Efficacy of Adoptive Immunotherapy Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The Ame[.]

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TARGETING GENE MODIFICATION AND INTEGRATION

Adenovirus with an Arginine-Grafted Bioreducible

Polymer on Transduction Ef ciency and

Immunogenicity in Cancer Gene Therapy

Pyung-Hwan Kim,1,2 Tae-il Kim,3 James W Yockman,3 Sung Wan

Kim,3,4 Chae-Ok Yun.1,2

1 Graduate Program for Nanomedical Science and Technology,

Yonsei University, Seoul, Republic of Korea; 2 Brain Korea 21

Project for Medical Sciences and Institute for Cancer Research,

Yonsei University College of Medicine, Seoul, Republic of Korea;

3 Department of Pharmaceutics and Pharmaceutical Chemistry,

Center for Controlled Chemical Delivery, University of Utah,

Salt Lake City, UT; 4 Department of Bioengineering, College of

Engineering, Hanyang University, Seoul, Republic of Korea.

Adenoviral vectors have been widely used for cancer gene therapy

due to many advantages, such as high transduction ef ciency, but

safety concerns related to severe immunogenicity and other side

effects have led to careful reconsideration of their use in human

clinical trials To overcome these issues, a strategy of generating

hybrid vectors that combine viral and non-viral elements as more

intelligent gene carriers has been employed Here, we coated

adenovirus (Ad) with a novel arginine-grafted bioreducible polymer

(ABP) via electrostatic interaction We examined the transduction

effect of ABP-coated Ad complex at various ABP molecules/

Ad particle ratios We also examined the coating of Ad with ABP

polymers at the optimal polymer ratio using dynamic light scattering

and transmission electron microscopy and con rmed the coating

by NaCl treatment because the slat ions of NaCl is more strong ion

interaction than that of ABP to virus The characterization of complex

was compared the size and surface charge of ABP-coated Ad complex

formed by physical interaction compared with naked Ad by Zetasizer

In both high and low coxsackie virus and adenovirus receptor

(CAR)-expressing cells, ABP-coated Ad complex produced higher levels

of transgene expression than cationic polymer 25K PEI Notably,

high cytotoxicity was observed with 25K PEI-coated Ad complex

treatment In addition, ABP-coated Ad complex was not signi cantly

inhibited by serum, in contrast to naked Ad Moreover, ABP-coated

Ad complex signi cantly reduced the innate immune response

relative to naked Ad, as assessed by interleukin-6 (IL-6) cytokine

release from macrophage cells Overall, our studies demonstrate

that the combination of Ad with ABP polymer offers the potential

to increase the ef ciency of vectors for gene therapy by shielding

the virus from deactivation by the immune system, and may make

systemic administration feasible

Targeting Gene Modi cation and Integration

87 Safe and Ef cient Delivery of

Meganucleases for Lentivirus-Mediated Gene

Targeting

Araksya Izmiryan,1 Alix Bourdel,1,2 Arnaud Jollet,1 Frédéric

Pâques,2 Olivier Danos.1

1 Université Paris Descartes, INSERM U781, Paris, France;

2 Cellectis SA, Romainville, France.

Gene therapy treatments using retroviral and lentiviral vectors have

revealed the genotoxicity associated with random insertion of vector

DNA into the genome Our goal is to design lentiviral vectors with

which a therapeutic sequence is inserted at a universal safe locus in the

human genome, using homologous recombination (HR)

Lentiviral-mediated HR occurs at high frequency when a DNA recombination

matrix is delivered to cells along with a site speci c endonuclease that

creates a locus-speci c double strand break Heterodimeric Zinc  nger

Nucleases as well as single chain meganucleases can be used for this

purpose However, for therapeutic applications, it is preferable to have

an appropriate control of the amount of endonuclease in the target cells, and especially, to avoid sustained expression One possibility

is to vectorize the protein itself, instead of an expression cassette

Here we describe experiments showing that highly ef cient HR is obtained when the nuclease is delivered as a protein incorporated

in the lentiviral particles I-SceI, a prototypic meganuclease from yeast, was incorporated into the virions as a fusion with Vpr, an HIV accessory protein Non-integrating lentiviral vectors (NILV) containing a recombination matrix and the I-SceI fusion protein were produced at high titers We have used a CHO-derived reporter cell line in which I-SceI mediated HR events result in the repair of

a puromycin resistance gene These cells were transduced by NILVs containing both I-SceI as a Vpr fusion and the recombination matrix,

or by separate vectors encoding either the recombination matrix or I-SceI Puromycin resistant cell clones were selected and analysed

by PCR and Southern blot for targeting events A majority of puromycin resistant clones contained the expected genomic structure and globally, our data indicate that the transfer of the endonuclease

as a virion incorporated protein results in up to 5% of HR events,

in a dose dependent manner HR levels were lower when the fusion protein and the recombination matrix were provided in separate viral particles, suggesting that the physical association of the two in the same lentiviral pre-integration complex may be important for ef cient targeting Interestingly, the levels of HR obtained when I-SceI was encoded by the lentiviral vector were consistently lower Finally, we have shown that Vpr fusions could also be used for the transduction

of a single chain meganuclease (scMN) engineered from I-CreI with

a recognition site in the human Rag-1 gene We are now using this system with various scMN designed to target transgene insertion in candidate “safe havens” on the human genome

Human Primary T Lymphocytes To Improve the Safety and Ef cacy of Adoptive Immunotherapy

Pietro Genovese,1,2 Elena Provasi,3,2 Angelo Lombardo,1,2 Zulma Magnani,3 Andreas Reik,5 Pei-Qi Liu,5 Oscar Muniz Pello,1 Jurgen Kuball,4 Philip D Gregory,5 Michael C Holmes,5 Philip D

Greenberg,4 Chiara Bonini,3 Luigi Naldini.1,2

1 HSR-TIGET, Milan, Italy; 2 San Raffaele University, Milan, Italy;

3 San Raffaele Scienti c Institute, Milan, Italy; 4 Fred Hutchinson Cancer Research Center, Seattle; 5 Sangamo BioSciences, Richmond, CA.

Editing the genetic content of clinically relevant primary cells, such as T lymphocytes, has long been a major goal for gene therapy

Indeed, development of an ef cient method for gene targeting in these cells can overcome many of the technological and safety issues of the current gene replacement strategies for adoptive immunotherapy

Here, we present a novel approach based on Zinc Finger Nucleases (ZFN) and Integrase Defective Lentiviral Vectors (IDLV) for: i) targeted integration of transgenes and ii) site-speci c gene knock out in primary human T cells First, we developed a platform for site-speci c integration into two putative safe genomic harbors, the

CCR5 gene and the AAVS1 locus Codelivery of ZFNs targeting

either site by Adenoviral vector (Ad5/F35) in association with IDLV containing a GFP expression cassette  anked by locus homology arms, resulted in ef cient site-speci c integration (6%) Molecular analysis on GFP+ clones con rmed site-speci c integrations at the ZFN targeted sites with disruption of the sister allele by NHEJ in

up to 87% of clones GFP expression was higher from the AAVS1

than the CCR5 site Moreover, gene expression analyses showed

that integration of an expression cassette into AAVS1 did not perturb expression of  anking genes To test the feasibility of this approach

in a therapeutically relevant setting, we transferred exogenous T cell receptor (TCR) genes ZFN-mediated integration of a high-avidity

resulted in ∼18% gene targeting Next, exploiting ZFNs to create gene knockouts we addressed a major hurdle with TCR gene transfer, the co-expression of endogenous and tumor speci c TCRs in the same cell The expression of 4 TCR chains not only diminishes expression

of the desired TCR, due to competition for CD3 molecules, but also leads to TCR mispairing, which results in unpredictable speci cities that can be autoreactive Thus, we developed ZFNs speci c for the

constant regions of TCR β chain gene (TRBC1 and TRBC2) and

exploited the NHEJ-repair to disrupt the endogenous TCR ZFN treatment resulted in functional inactivation of this gene in up to 7% of primary T cells, as indicated by generation of cells that do not express the CD3/TCR complex on cell surface, in the absence of evident toxicity Molecular analyses revealed equal NHEJ mediated gene disruption of both TRBC loci LV-mediated transfer of the WT1-TCR was ef ciently achieved in CD3neg sorted cells (>45%) and resulted in higher expression of the tumor speci c TCR than that observed with conventional gene transfer Overall, our results demonstrate that both ZFNs mediated gene addition and gene disruption can be successfully combined in T lymphocytes to facilitate rapid generation of effective and safe T cells for adoptive immunotherapy *equal contribution

Recombination with Less Toxicity Than Double-Strand Breaks Using an AAV Template

Michael J Metzger,1 Audrey McConnell-Smith,2 Barry L

Stoddard,2 A Dusty Miller.1

1 Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; 2 Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA.

The enhancement of homologous recombination (HR) using targeted double-strand breaks (DSBs) has the potential to correct gene defects in their endogenous loci, avoiding many problems which have plagued traditional gene therapy However, even a perfectly site-speci c DSB is a DNA damaging event, and resulting toxicity and potential for mutagenesis and translocations are serious problems for this strategy We compared the ability of nicks and DSBs to enhance homologous recombination using the homing endonuclease I-AniI and a redesigned variant which produces only single-strand nicks When both template and I-AniI expression plasmids were transfected into 293 cells containing an integrated copy of an inactive

lacZ target, we showed that the nickase enhances HR up to 300-fold

above transfection of template plasmid alone (compared to 8,000-fold enhancement with the DSB-inducing enzyme) When we delivered the template with an AAV vector and the endonuclease construct with a lentivirus for longer expression, we also found that both DSBs and nicks enhance HR in a manner dependent on the amount

of endonuclease used While HR was induced with lower amounts of DSB-inducing enzyme than with nick-inducing enzyme, the toxicity observed with the DSB-inducing enzyme was far more severe (>80%

cell death at an MOI of 1) In contrast, the toxicity of the nicking endonuclease was low and was not distinguishable from the toxicity

of an inactive endonuclease or the toxicity of an empty lentivirus vector expressing only the mCherry marker used to titer vectors

Due to this DSB toxicity, the maximum amount of HR observed with nicks and with DSBs was similar (20-60 nick-induced foci compared

different lacZ target sites were investigated: one in which the 19 bp I-AniI recognition site replaced a 19 bp region in the lacZ gene and

the other in which the I-AniI site was inserted into the same location

Interestingly, the lacZ target with the “replacement” inactivating

mutation supported nick-induced HR at a 10-fold higher rate than the target with the “insertion” mutation in both the AAV assay and the transfection assay, while no difference in DSB-induced HR was observed between the two targets It has been suggested that nicks could only stimulate HR upon conversion to a DSB, making nicks

simply a less ef cient version of DSB-induced HR; however, these results suggest that is incorrect Both the observation that nicks can stimulate HR with lower toxicity than DSBs and the observation that target site design effects nick-induced HR but not DSB-induced HR strongly argue that nicks induce HR through a different mechanism than DSBs Therefore, this strategy of HR with a nicking enzyme and a viral delivery system may be useful in clinical gene therapy applications, allowing for more ef cient gene correction without the toxicity and mutagenic activity of DSBs

Chromosomes Using a Homing Endonuclease

Byoung Y Ryu,1,2 Michael T Certo,1,2 Mikhail Garibov,1,2 Andrew

M Scharenberg,1,2 David J Rawlings.1,2

1 Northwest Genome Engineering Consortium (NGEC), Seattle, WA; 2 Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA.

Homing endonucleases (HE) are a class of rare cutting nuclease that recognize and cleave 19-22 base pair (bp) sequences with a limited degree of degeneracy They represent an emerging tool for genome engineering as they can be used to induce site speci c DNA double strand breaks (DSB) and initiate homologous recombination or gene insertion Among known HE, I-AniI recognizes 19 bp asymmetric sequences, and has no exact match in human genomic sequence databases To determine if I-AniI can induce DSB in cells, we performed BLAST search to the known I-AniI target site to identify near-cognate matches in the human genome potentially susceptible

to cleavage A set of 60 near-cognate sites were individually cloned into reporter vectors in order to compare rates of in cellulo cleavage to levels of cleavage predicted via in vitro pro ling Three near-cognate sites were identi ed that cleaved with high ef ciency in the reporter assay including: one intronic, one exonic, and one intergenic region site To evaluate the accessibility of these sites within genomic DNA, integration de cient lentiviral vectors (IDLV) were developed to express I-AniI variants in human pre-B cell line, Nalm-6 cells As DSB are frequently repaired through non-homologous end joining (NHEJ) pathway, the rate of genomic NHEJ events after IDLV transduction was used as surrogate marker for cleavage ef ciency Consistent with in cellulo cleavage, all three near-cognate sites exhibited similar NHEJ rates ranging from 15 to 20% The percentage

of NHEJ events was even higher (>70%) when integrating LV were used to stably express I-AniI in human cells supporting the conclusion that I-AniI can recognize and cleave these sites in vivo The intergenic locus was also evaluated as a site for targeted gene insertion When a GFP expression cassette (1.8 kb) with  anking homologous sequences was introduced into human Nalm-6 cells along with I-AniI, an estimated 1% of targeted GFP gene insertion into this site was observed in an initial screening of single cell clones The ef ciency of HE-induced targeted gene insertion in other sites is presently under evaluation

Facilitates rAAV-Mediated Gene Targeting

Yonglun Luo,1,3 Emil Kofod-Olsen,2 Jenny Blechingberg,1 Rikke Christensen,1 Nicklas Heine Staunstrup,1 Lars Bolund,1,3 Charlotte Brandt Sørensen.1

1 Department of Human Genetics, Aarhus University, Aarhus C, Denmark; 2 Department of Medical Microbiology and Immunology, Aarhus University, Aarhus C, Denmark; 3 Hua Da/BGI, Shenzhen, China.

Recombinant adeno-associated virus (rAAV) mediated gene targeting greatly facilitates homologous recombination (HR) compared to plasmid DNA methods However, it is still hampered

by low targeting and high random integration frequency It has been

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