545 regulation of AAV mediated transgene expression in airway epithelium in vivo

545 Regulation of AAV Mediated Transgene Expression in Airway Epithelium In Vivo Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S209 GENE R[.]

Molecular Therapy Volume 19, Supplement 1, May 2011

GENE REGULATION II

AAV2/5-mediated EGFP expression to the RPE, while the inclusion

of miR204Ts restricted transgene expression to PRs, as expected

In addition, the use of miR204Ts effi ciently restricted the

AAV2/8-mediated expression of AIPL1- a gene mutated in Leber congenital

amaurosis, a common form of childhood inherited blindness- to

porcine PRs In conclusion, we show that miRNA-based regulation

of transgene expression can be applied in the retina to restrict the

robust expression levels obtained using ubiquitous promoters to a

specifi c cell type Alternatively, it can be coupled with cell-specifi c

promoters thus providing an additional layer of fi ne-tuning transgene

expression

543 Creation of Structural Variations in the

Human Genome Using Engineered Zinc Finger

Nucleases

Eunji Kim,1 Hyung Joo Lee,1 Jin-Soo Kim.1

1 Department of Chemistry, Seoul National University, Seoul,

Korea.

Many genetic diseases and cancer are associated with chromosomal

rearrangements such as deletions, insertions, duplications,

translocations, and inversions Even among healthy individuals,

thousands of different structural variations (SVs) or copy number

variations resulting from different chromosomal rearrangements are

observed Despite the recent discoveries of and interest in numerous

structural variations (SVs) in the human and other higher eukaryotic

genomes, little is known about the etiology and biology of these

SVs, partly due to the lack of molecular tools with which to create

individual SVs in cultured cells and model organisms Here, we

present a novel method of inducing targeted genome rearrangements

using engineered zinc fi nger nucleases (ZFNs) We found that ZFNs

designed to target two different sites in a human chromosome could

introduce two concurrent double-strand breaks, whose repair via

non-homologous end-joining (NHEJ) gives rise to targeted deletions,

duplications, and inversions of the genomic segments of up to a mega

base pair in length between the two sites We also found that ZFNs can

induce incorporation of synthetic ODN cassettes into the targeted site

and replace the cassettes with the large chromosomal DNA segments

High frequencies of ZFN-induced genomic rearrangements, ranging

from 0.01% to 12%, allowed us to routinely isolate clonal populations

of cells whose genomes contain SVs of interest We propose that

ZFNs can be employed as molecular tools to study mechanisms of

chromosomal rearrangements and to create SVs in a predetermined

manner so as to study their biological roles In addition, our method

opens up new possibilities for correcting genetic defects caused by

chromosomal rearrangements and, thus, holds promise in gene and

cell therapy

Neuroprotective in Rodent Preclinical Models of

Stroke

S Kaye Spratt,1 Philippe M D’Onofrio,2 Mark M Magharious,2,3

Mahinthan Thayapararajah,2 Richard Surosky,1 Gary Lee,1 Martin

Giedlin,1 Dale Ando,1 Michael Fehlings,3 Paulo D Koeberle.2,3

1 Development, Sangamo BioSciences Inc, Richmond, CA; 2 Dept

Surgery, University of Toronto, Toronto, ON, Canada; 3 Department

of Genetics and Development, Toronto Western Research Institute,

and Spinal Program, Krembil Neuroscience Centre, University

Health Network, Toronto, ON, Canada.

Vascular endothelial growth factor (VEGF) is most widely

recognized for its role in angiogenesis; however recent studies have

identifi ed anti-apoptotic and neuroprotective functions for VEGF in

the CNS VEGF therapy for CNS injury has been hampered by the

tendency of VEGF to promote vascular permeability, edema, and

immune infi ltration into tissues Recently, engineered zinc fi nger

proteins that upregulate multiple isoforms of VEGF-A (VEGF-ZFP) in their natural biological ratios have been developed and this approach is promising because VEGF upregulation by ZFPs does not promote vascular permeability (Rebar et al, 2002) Highly reproducible models of optic nerve transection (axotomy), ophthalmic artery ligation and a pial strip traumatic injury model were used to determine if the VEGF-ZFP is neuroprotective in the adult CNS AAV2 vectors encoding VEGF ZFPs (AAV.ZFP) or control vectors encoding GFP (AAV.GFP) were delivered by intraocular injections

3 days prior to retinal injury Retinal ganglion cells (RGCs) were retrogradely labeled with Fluorogold in order to quantify survival from fi xed fl atmounted retinas AAV.ZFP treatment increased RGC survival by 2-fold (n=6/group, p<0.01) after optic nerve transection Similarly AAV.ZFP treatment signifi cantly enhanced RGC survival

at 14 or 21 days after ophthalmic artery ligation (n=6/group, p<0.01) Function of the retino-tectal pathway was assessed by pupillometry at

7, 14, or 21 days after retinal ischemia, and AAV.ZFP treated retinas demonstrated signifi cantly greater pupillary responses than controls

at all time points tested RECA-1 immunostaining demonstrated no appreciable differences between retinas treated with AAV.ZFP or controls, suggesting that AAV.ZFP treatment did not affect retinal vasculature Following pial strip injury of the forelimb cortex, brains treated with an adenovirus encoding the VEGF ZFP (Ad.ZFP) showed higher neuronal survival (n=5, p<0.05) adjacent to the devascularized infarct and accelerated wound contraction between 1 and 4 weeks after injury compared to control animals receiving Ad.GFP Behavioral testing using the cylinder test for vertical exploration demonstrated that Ad.ZFP treated animals had significant improvements in contralateral forelimb function within the fi rst two weeks after injury (n=8, p<0.05) than controls at week 1 (post-stroke) through

to week 6 (post-stroke) The recovery was most pronounced early after injury Our results demonstrate that delivery of the VEGF-ZFP activator following retinal and ischemic injury in preclinical models

of stroke is neuroprotective Improvements in retinal ganglion cell survival, signifi cantly greater pupillary responses and short-term improvements in forelimb function were observed These results suggest the therapeutic potential of ZFPs for the treatment of cerebra-vascular insults in CNS trauma and stroke

Expression in Airway Epithelium In Vivo

Maria P Limberis,1 Melissa Fontana,2 ShuJen Chen,1 James M Wilson.1

1 Gene Therapy Program, Department of Pathology & Laboratory

Medicine, University of Pennsylvania, Philadelphia; 2 University of the Sciences in Philadelphia, Philadelphia College of Pharmacy, Philadelphia.

Gene transfer vectors based on the adeno-associated virus (AAV) have been shown to effectively transduce cells of the nasal and lung airway epithelium in mice and macaques When engineering viral-based vectors as therapeutics for genetic lung diseases, persistent gene expression is necessary to maintain the therapeutic effect To this vein, transgene expression for genetic lung diseases that include cystic fi brosis and α-1-antitrypsin defi ciency is typically driven

by constitutive AAV vectors For disease states in which transient transgene expression is required, the regulation of vector-mediated gene expression is warranted To regulate gene expression in airway cells in vivo we used an AAV-based inducible vector system that

is activated by rapamycin We previously showed that AAV2/9 effi ciently transduces the respiratory epithelium that lines the mouse nasal airways To test the performance of the inducible AAV vector system in airway, we intranasally co-injected 1011 genome copies

of the inducible AAV2/9 vector expressing fi refl y luciferase (ffLuc) and transcription factor in both nares of 6-8 wk-old C57Bl/6 male mice (n=5/group) Control mice included those injected with a) the

Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy

S210

GENE REGULATION II

inducible AAV2/9 vector expressing ffLuc; b) the AAV2/9 vector

expressing transcription factor; and c) the constitutive AAV2/9 vector

expressing ffLuc (used to assess the maximally achievable ffLuc

expression) Twenty-one days after vector injection, the degree of

gene expression ‘leakage’ of the inducible AAV vector system as well

as the level of ffLuc in mice injected with the constitutive AAV2/9

vector were assessed Briefl y, luciferin was delivered intranasally

and using the Xenogen IVIS imaging system all control and

vector-treated mice were imaged Only mice injected with the constitutive

AAV2/9 vector expressed ffLuc (∼1.6 x106 photons/seconds; p/s) at

levels signifi cantly above baseline (103 p/s) No “leakage” of ffLuc

from the inducible AAV vector system was observed All mice were

then injected intraperitoneally with 1mg/kg rapamycin and ffLuc

expression was monitored daily (for up to 7 days) Twenty-four hours

later we observed a ∼1000-fold increase in ffLuc expression in the

nasal airways of mice injected with the inducible AAV vector system

Indeed the level of ffLuc expression (∼1.6 x106 p/s) was statistically

no different to that conferred by the constitutive AAV2/9 vector (P<

0.05, ANOVA, SNK test) ffLuc expression for the control groups

remained similar to baseline We observed a modest increase in ffLuc

expression in those mice injected with the constitutive AAV vector

The induction was repeated three weeks later and similar levels of

increase in ffLuc expression were observed No adverse events to

either of the two rapamycin inductions were observed in any of

the treated mice Here we demonstrate the utility of an AAV-based

inducible vector system designed to regulate gene expression in

airway epithelium in vivo, which is particularly applicable to disease

states that require short-term high levels of gene expression

New Tet-ON AAV Vector for Tightly Controlled

Transgene Expression

Davide Gianni,1 Nancy H Tran,2 Dimphna H Meijer,2 Miguel

Sena-Esteves.1

1 Neurology and Gene Therapy Center, University of Massachusetts

Medical School, Worcester, MA; 2 Neurology and Neuroscience

Center, Massachussetts General Hospital, Charlestown, MA.

The regulation of transgene expression is a critical aspect for

gene therapy-based therapeutic approaches In particular this aspect

is of primary importance when the genes encoded in the vectors

can potentially interfere with the cell metabolism and survival

Tetracycline (Tet)-mediated regulation of gene expression has been

extensively investigated and several generations of Tet-responsive

systems have been developed However most of the systems in the

context of viral vectors are affected by signifi cant basal expression in

the non-induced state (OFF state) Here we describe a new AAV vector

system (TetD-AAV) carrying two different Tet-responsive elements

(rtTA2s–M2 activator and tTSKid repressor) in cis with the transgene

in order to achieve low background expression in OFF state and

sustained expression during induction The in vitro characterization of

gene expression regulation was performed with a TetD-AAV2 vector

encoding Gaussia luciferase (Gluc) as reporter gene The vector was

tested on several cell lines including 293T, U87, GL261, SH-S5Y5 and

normal human astrocytes transduced at vector doses of 1e5 genome

copies (gc)/cell Gluc assays in the supernatant of transduced cells

showed low Gluc levels in the OFF state and expression levels in the

ON state comparable to those obtained with an AAV vector encoding

Gluc under a CBA promoter and also carrying a WPRE element This

resulted in induction ratios of 400- to 1200-fold depending on the cell

line with maximum transgene expression observed at a doxycycline

(Dox) concentration of 1 µg/ml Additional in vitro characterization

was performed replacing Gluc with human (h) or murine (m)

interferon b (IFN-b) in the vector and verifying the effects of these

cytokines on the growth kinetics of glioblastoma cell lines U87 and

GL261 in culture Our results clearly showed signifi cant inhibition

of tumor cell growth in the presence of Dox (p<0.001), but minimal effects in the absence of Dox (OFF state) As the IFN-b anti-tumoral effects are species-specifi c, we transduced U87 and GL261 cells with TetD-AAV2-mIFN-b and TetD-AAV2-hIFN-b, respectively, at 1e5 gc/ cell to quantify IFN-b levels in culture medium in the ON and OFF states using ELISA assays Our results showed that in the presence of Dox the TetD-AAV2-IFN-b vectors expressed IFN-b levels 2-10-fold lower that those obtained with AAV2 vectors encoding IFN-b under

a constitutive promoter, but extremely low levels in the absence of Dox (<detection limit for hIFN-b produced in GL261) In conclusion

we have developed a new Tet-regulated AAV vector characterized by

tight regulation of transgene expression in cultured cells Ongoing in

vivo studies will characterize the regulation characteristics of the new

TetD-AAV vector by bioluminescence imaging after intraparenchymal infusion in the adult mouse brain

Suppresses Mechanical Allodynia Induced by Mononeurpathic Pain in Rats

Chung-Ren Lin,1,2 Kuan-Hung Chen,1 Chih-Hsien Wu,1 Yi-Shen Chen.1

1 Anesthesiology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan; 2 Anesthesiology, National Taiwan University Hospital, Taipei, Taiwan.

BACKGOUND: Neuropathic pain is a pain that comes from problems with signals from the nerves It is characterized by spontaneous pain, thermal hyperalgesia and mechanical allodynia and often occurs as a result of injury to peripheral nerves To understand the molecular mechanisms underlying neuropathic pain,

it is essential to elucidate how nerve injury alters gene expression and how the change contributes to the development and maintenance of chronic pain MicroRNAs regulate gene expression in a wide variety

of biological processes mainly at the level of translation Recent studies have suggested that miRNA might play a role in neuropathic pain METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the role of miR-183 in a rat spinal nerve ligation (SNL) model and explored the feasibility of treating neuropathic pain by replacing miR-183 Our results demonstrated that expression of miR-183 decreased signifi cantly in SNL DRG neurons compared

to controls Intrathecal lentivirus-mediated delivery of miR-183 attenuated SNL-induced mechanical allodynia might through inhibiting the activation of Nav1.3 and BDNF CONCLUSIONS/ SIGNIFICANCE: These fi ndings suggest that replacement of

miR-133 may be a new strategy for the treatment of neuropathic pain

548 Differential Regulation of Multiple Transgenes by Alternative Splicing

Eun-Young Choi,1 Xiaohuai Zhou,1 Richard J Samulski.1

1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.

Regulation of transgene expression is necessary in certain gene therapy strategies to achieve optimal results and avoid adverse effects from over-expression, especially given the long-term retention of the transgene in the target cells In this regard, we are developing

a novel regulation system to control transgene expression mediated

by adeno-associated virus (AAV) vectors, which can provide long-term transduction of host cells This regulation system is based on the alternative splicing mechanism of the mutant human b-globin intron-2, IVS2-654 We incorporated IVS2-654 into expression cassettes and demonstrated that the expression of the transgenes could be induced by the application of an antisense oligonucleotide (ASO) corresponding to the 5’ splice site of the intron, leading to alternative splicing Furthermore, we generated an optimized