127 PROGNOSE: an online tool for predicting and analyzing the off target site effects of designer nucleases

127 PROGNOSE An Online Tool for Predicting and Analyzing the Off Target Site Effects of Designer Nucleases Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene[.]

Molecular Therapy Volume 20, Supplement 1, May 2012

DNA VECTOR DEVELOPMENT & GENE TARGETING I

attributed to expression of HAC-encoded genes This generation of

human artifi cial chromosomes should be suitable for studies of gene

function and therapeutic applications

of Plasmids Encoding Pathogenic Antigen- and

Cytokine-Genes

Yoshiyuki Koyama,1 Chieko Yoshihara,1 Tomoko Ito,1,2 Masazumi

Eriguchi.3

1 Otsuma Women’s University, Tokyo, Japan; 2 Musashino

University, Nishitokyo City, Japan; 3 Shin-yamanote Hospital,

Higashi-Murayama City, Japan.

Although several viral vectors harboring cytokines have been

successfully employed to induce tumor-regression, non-viral vectors

having the same cytokine genes showed lower therapeutic effi cacy

One possible reason for this limited effi cacy would be low transfection

effi ciency of the non-viral systems However, it may be diffi cult to

induce immune response to tumor cells with low immunogenicity

only by cytokine-production Expression of the viral antigenic

proteins on the infected tumor cell surface would be essential to

stimulate APCc Not only the virus infection, but transfection of the

virus- or bacteria-specifi c antigenic protein genes could also induce

the pathogenic antigen presentation on the tumor cell surfaces

Simultaneous transfection with plasmids expressing cytokine and

pathogenic antigen into tumor cells should, thus, be expected to

lead to highly effective anti-tumor immune-enhancement Here,

we employed two pathogenic proteins, adenovirus death protein

(ADP) and mycobacterium tuberculosis early secretory antigenic

target-6 protein (ESAT-6) as an immune response-inducing antigen

As a carrier of these gene, we have developed a highly effective

gene transfection systems comprising very small (< 70 nm) plasmid

complexes with negative surface charge (Biomaterials 31 (2010)

2912–2918) In this study, the small complexes were made of the

plasmids harboring the pathogenic protein genes Those having

GM-CSF or IL-2 genes were also prepared Co-transfection of the

cytokine-gene with the pathogenic antigen-gene showed very high

anti-tumor therapeutic effect in tumor-bearing mice Animal clinical

study on primary tumor-bearing dogs was also carried out, and evident

suppression of the tumor growth was observed

Transgenesis in Human Cord-Derived Cells: En

Route to Cell Therapy for Hemophilia A

Jaichandran Sivalingam,1,2 Toan Thang Phan,3,4 Oi Lian Kon.1,2

1 Laboratory of Applied Human Genetics, Humphrey Oei Institute

of Cancer Research, National Cancer Centre, Singapore,

Singapore; 2 Department of Biochemistry, Yong Loo Lin School

of Medicine, National University of Singapore, Singapore;

3 Department of Surgery, Yong Loo Lin School of Medicine,

National University of Singapore, Singapore.

The inability of current gene therapy vectors to integrate transgenes

precisely into safe genomic sites has been associated with serious

adverse events in clinical trials Zinc fi nger nucleases (ZFNs) have

emerged as a potentially safer technique for targeted gene integration

ZFNs are chimeras of sequence-specifi c DNA-binding zinc fi nger

peptides fused to the catalytic domain of FokI endonuclease

They induce highly site-specifi c genomic incisions that enhance

homologous recombination-mediated integration of an exogenously

provided transgene (donor DNA) We have used ZFNs to integrate

a hybrid FVIII cDNA into the human AAVS1 locus Our goal is to

achieve durable constitutive secretion of FVIII from primary human

umbilical cord-lining epithelial cells (CLECs) with the intent of

developing them as autologous bioimplants for hemophilia A therapy

A pair of codon-optimised four-fi nger obligate heterodimeric ZFNs

(Miller JC, et al Nat Biotechnol 2007; 25:778) targeting the AAVS1

locus was assembled and expressed from a dual expression cassette plasmid vector N496D and H537R mutations were introduced into

the FokI domains of left and right ZFNs, respectively, to restore catalytic activity of obligate heterodimeric ZFNs (Doyon Y, et al Nat

Methods 2011; 8:74) S418P and K441E mutations were introduced

into both ZFNs to enhance cleavage activity (Guo J, et al J Mol Biol

2010; 400:96) Expression of optimized ZFNs in concert with ZFN-stabilising effects of mild hypothermia resulted in a 4.6-fold increase

in targeted integration of a 50-bp donor DNA in K562 cells (Student’s

t-test, p=0.001) Under optimized conditions of ZFN expression,

we have achieved integration of a 9-kb hybrid FVIII DNA cassette into the AAVS1 locus of K562 cells and primary CLECs Junctional PCR and sequencing showed precise insertion without evidence of indels at both transgene-genome junctions We have incorporated

a codon-optimized HSV TK in the donor DNA as a supplementary mechanism to minimize random transgene integrations A B-domain deleted human-porcine hybrid FVIII cDNA comprising porcine A1 and A3 domains was assembled in our lab with 93% identity

to human FVIII cDNA Compared to human FVIII cDNA, hybrid

FVIII cDNA increased in vitro FVIII secretion by transfected CLECs by 5-fold (Student’s t-test, p=0.003) The biosafety of

ZFN-modifi ed FVIII-expressing CLECs will be evaluated for alterations

in the transcriptome, genome copy number, chromosomal structures and for acquired tumorigenic potential in immunocompromised mice The presence of unintended off-target genome modifi cations will be investigated by whole genome sequencing of clonal cells Comprehensive assessment of low genotoxic risk coupled with

therapeutically meaningful and durable FVIII secretion in vivo could

justify developing transgenic CLECs as autologous cell therapy for hemophilia A

and Analyzing the Off-Target Site Effects of Designer Nucleases

Eli J Fine,1 Charles L Zhao,1 Thomas J Cradick,1 Gang Bao.1

1 Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, GA.

Designer nucleases are engineered restriction enzymes created

by fusing a DNA binding domain to a non-specifi c DNA cleavage domain When designer nucleases cleave a target locus, cellular DNA repair can be used to knockout or edit a gene, thereby facilitating therapeutic gene correction or the creation of genetically modifi ed model organisms Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) are major classes of engineered nucleases that are deployed in pairs, greatly increasing their specifi city Although each pair of nucleases is designed to target a unique genomic sequence, there have been a number of reports of ZFNs and TALENs cleaving additional genomic sites Off-target cleavage may signifi cantly impact the effi cacy and safety

of biomedical applications of designer nucleases We have developed, PROGNOSE (Predicted Report Of Genome-wide Nuclease Off-Site Effects), an online tool that performs exhaustive searches of genomic DNA sequences for potential nuclease off-target cleavage sites Different genomes can be searched based on homology to the nuclease pair’s target DNA binding sequences and user-supplied criteria, including the allowed spacings between nuclease binding sites, and the number of mismatches allowed in each binding domain PROGNOSE provides a list of possible off-site cleavage locations and annotates each site as being in an exon, promoter, intron, or intergenic region This information can be used while designing nuclease pairs to help identify target sequences without signifi cant homology to other sites in the genome For each potential off-target site identifi ed, PROGNOSE also automatically generates fl anking PCR primers allowing amplifi cation for quantifi cation of non-specifi c

Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy

S52

DNA VECTOR DEVELOPMENT & GENE TARGETING I

cleavage and misrepair events at these genomic loci using nuclease

assays or deep sequencing To validate the tool, we used PROGNOSE

to analyze off-target site cleavage events after transfecting pairs of

TALENs and ZFNs that target the same gene segment We performed

PCR amplifi cation and mutation detection assays to quantify the

level of misrepair from cleavage at the intended nuclease target

sites and putative off-target sites We compared these results to the

predicted off-target sites for both TALENs and ZFNs In summary,

PROGNOSE is a user-friendly online tool that makes the analysis

of nuclease off-target cleavage easily accessible, without the need

for bioinformatics and biochemical skill sets Presently off-target

effects of ZFNs and TALENs are poorly understood since only a

limited number of nuclease pairs have been tested As more

off-target site cleavage events are analyzed, the underlying trends that

dictate nuclease specifi city may become apparent This will greatly

aid the design and application of engineered nucleases in biological

and medical studies

B DNA Nuclear Targeting Sequence Improves In

Vitro Suicide Gene Therapy Effi cacy in Both Small-

and Non-Small Cell Lung Cancer Cell Lines

Frederik Cramer,1 Camilla L Christensen,2 Melissa Badding,3

David A Dean,3 Hans S Poulsen.1

1 Department of Radiation Biology, Copenhagen University

Hospital, Copenhagen, Denmark; 2 Dana-Farber Cancer Institute,

Boston; 3 Department of Pediatrics, University of Rochester.

The 5 year survival rate of small cell lung cancer (SCLC) is

currently 6% and new treatments are in high demand Gene therapy

could be a promising candidate for a novel treatment of SCLC To

target primary and metastatic tumor tissue systemic administered

treatment is required However non-viral systemic delivery of

therapeutic DNA is currently insuffi cent We therefore investigated

if gene therapy delivery could be improved by implementing a DNA

Nuclear Targeting Sequence (DTS) strategy To test this, multiple

Nuclear Factor kappa B (NFkB) binding sequences were cloned

into a transcriptional targeting plasmid encoding the yeast cytosine

deaminase (YCD) and yeast uracil phosphoribosyl transferase

(YUPRT) genes The expression of the suicide genes was regulated

by the SCLC-specifi c promoter Insulinoma-associated 1 (INSM1)

The therapeutic effects of the different plasmid constructs were

evaluated by a viability assay in vitro and the nuclear translocation

effect of plasmids by microscopy A positive correlation between the

number of inserted NFkB binding sites and the therapeutic effect were

observed in several SCLC cell lines The effect was most likely due to

elevated nuclear translocation of the suicide gene encoding plasmids

as we observed that 68% of SCLC cells microinjected with plasmids

containing NFkB binding sites, had nuclear localized plasmids 4 hours

post cytoplasmic microinjection When the experiment was repeated

with plasmids lacking the NFkB binding sites, the number of cells with

nuclear localized plasmids had dropped to 10 % The NFkB inserts did

not compromise the specifi city of the INSM1-promoter Furthermore,

the NFkB-inserts also resulted in a similar nuclear translocation

and cytotoxic effect in a non-small cell lung cancer (NSCLC) cell

line, indicating that therapeutic effi ciencies in other types of lung

diseases than SCLC could be obtained by the implementation of a

DTS strategy To our knowledge this is the fi rst time a DTS strategy

has been implemented in a suicide gene therapy system The results

show that a signifi cant improvement of gene therapeutic effi ciency

can be obtained by increasing the nuclear translocation of therapeutic

DNA in vitro We are currently testing the system in vivo.

Recombination-Mediated Genome Editing through Donor DNA Processing

Maarten Holkers,1 Manuel A F V Gonçalves.1

1 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, Zuid-Holland, Netherlands.

Current approaches to bring about efficient homologous recombination (HR)-mediated genome editing of human somatic cells are based on artifi cial hybrid nucleases designed to introduce double-stranded DNA breaks (DSBs) at predefi ned sequences of choice Recent research from our laboratory and those of others indicates that site-specifi c nicks or single-stranded DNA breaks (SSBs) provide for an alternative trigger for HR during gene targeting

or gene repair protocols (van Nierop et al [2009] Nucleic Acids Res 37: 5725-5736; McConnell-Smith et al [2009] Proc Natl Acad Sci

USA 106: 5099-5104; Metzger et al [2011] Nucleic Acids Res 39:

926-935) These SSB-assisted genetic modifi cation strategies have exploited either natural or engineered sequence- and strand-specifi c enzymes in the form of the adeno-associated virus (AAV) Rep78/68 proteins or a mutant I-AniI homing endonuclease, respectively Despite their differences in what the tools and the experimental set-ups are concerned, DSB- and SSB-mediated genome editing approaches have in common the fact that, hitherto, they have relied exclusively

on the endonucleolytic cleavage of the target chromosomal template Recently, using a cellular model based on AAV Rep78/68 proteins

and the human AAVS1 locus at position 19q13.42, we asked whether

introduction of SSBs in both donor and chromosomal target DNA molecules would enhance the rate of HR in human cells as posited by initial nick-based HR models We found that donor DNA processing via AAV Rep78/68-induced nicking greatly fosters HR-dependent

AAVS1 gene targeting (Gonçalves et al [2011] Nucleic Acids Res

Epub ahead of print) Here we hypothesized that recognition and processing of inverted repeats with the capacity to acquire

high-order conformations in vivo, by cellular structure-specifi c nucleases,

stimulate the use of donor DNA as DSB-repairing template To test this new gene targeting principle based on donor DNA processing, we generated and characterized cell lines containing a reporter expression unit whose open reading frame is disrupted by a premature stop codon plus the 18-bp I-SceI homing endonuclease target site Restoration

of reporter gene expression was shown to be strictly dependent on the presence of I-SceI and homologous gene-correcting exogenous DNA sequences Importantly, cell cultures exposed to wild-type I-SceI and homology-bearing templates equipped with secondary structure-forming sequences displayed a modest but nonetheless highly signifi cantly increase in the frequency of gene-corrected cells when compared to those incubated with I-SceI and conventional donor DNA (143%, p<0.0001) Our data suggest that the incorporation of high-order DNA-forming motifs in exogenous DNA templates might contribute to improved homology-directed gene repair protocols

Zinc-Finger Nuclease Mediated Gene Addition in Mouse Primary Fibroblasts

Jenny C Barker,1 Lily J Huang,2 Matthew H Porteus.3

1 Medical Scientist Training Program, UT Southwestern Medical Center, Dallas, TX; 2 Cell Biology, UT Southwestern Medical Center, Dallas, TX; 3 Pediatrics, Stanford University, Stanford, CA.

Nuclease-mediated safe harbor gene addition strategies are promising as next generation gene therapy technology One criterion that currently defi nes a “safe harbor” is that the locus can be disrupted without physiologic consequence In this abstract, we propose a modifi cation of homologous recombination-mediated safe harbor targeting that does not require disruption of the endogenous gene product In short, DNA that results in the same amino acid sequence