445 positive responses in a mouse model of leber congenital amaurosis after treatment with compacted DNA nanoparticles

445 Positive Responses in a Mouse Model of Leber Congenital Amaurosis after Treatment with Compacted DNA Nanoparticles Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Soci[.]

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Molecular Therapy Volume 18, Supplement 1, May 2010

by histopathology and ERG in eyes treated with ShHY10- GDNF

Moreover, this rescue persisted for a signi cantly longer duration (>5

months post-injection) than previous studies

The GDNF protein concentration in retinal homogenates and

vitreal  uid of injected animals was ∼2500 pg/ml, (10-fold greater

than previous studies of GDNF-induced rescue) Furthermore,

signi cant anatomic and functional improvement was observed

Neuroprotection of photoreceptors by GDNF overexpression from

MG is a highly promising strategy to slow retinal degeneration The

use of MG-speci c AAV from the vitreous is advantageous in terms of

safety, ef cacy, and longevity Therefore, the less invasive intravitreal

approach has potential for the treatment of retinal degenerations of

known and unknown etiology

443 AAV-Mediated Gene Supply of

Non-Erythropoietic Erythropoietin Derivatives Improves

Photoreceptor Morphology and Function in Models

of Retinal Degenerative Diseases

Pasqualina Colella,1,2 Carolina Iodice,1 Umberto Di Vicino,1

Alberto Auricchio.1,3

1 Telethon Institute of Genetics and Medicine (TIGEM), Naples,

Italy; 2 The Open University, Milton Keynes, United Kingdom;

3 Medical Genetics, Dept of Pediatrics, Federico II University,

Naples, Italy.

Gene supply of neurotrophic/antiapoptotic factors provides a

mutation-independent therapy for inherited retinal degenerations

(IRDs) that is desirable given their high genetic heterogeneity

In addition photoreceptor cells preservation achieved by gene

supply may allow subsequent gene-speci c therapy The cytokine

Erythropoietin (EPO) has shown promising neurotrophic and

anti-apoptotic effects in several models of neuronal degeneration including

IRDs as we have previously reported that systemic adeno-associated

viral (AAV) vector-mediated EPO delivery protects animal models

from photoreceptor degeneration Translation of these findings

into therapeutic application looks promising, however the EPO

erythrodifferentiating function causes several undesired side effects

Recently, EPO derivatives that do not increase hematocrits but retain

tissue-protective and anti-apoptotic functions have been identi ed

We are currently investigating the potential neuroprotective effects

of the EPO mutant S100E (EpoS100E) and of 2 short EPO-derived

peptides following AAV2/1 intramuscular and intraocular delivery in

animal models of light-induced and inherited retinal degenerations

High EpoS100E levels were measured in the sera and ocular  uids of

albino rats administered systemically and subretinally, respectively

Rats treated with EpoS100E but not with the Epo peptides had a minor

increase in hematocrits After light-damage, animals administered

either systemically or intraocularly with AAV1-CMV-EpoS100E

or-Epo peptides showed increased photoreceptor survival and preserved

retinal function although to a lesser extent compared to wild type EPO

Systemic delivery of Epo or EpoS100E or intraocular delivery of Epo

peptides were most effective We are currently testing EpoS100E and

EPO-derived neurotrophic peptides in the Aipl1-/- and Phrp2rd2/rd2

animal models of IRDs

In conclusion, our data support the neuroprotective effect of

non erythropoietic Epo derivatives in retinal degenerations while

con rming an important role of EPO erythropoietic activity in

photoreceptor protection

444 Early Indicators of Biological Ef cacy in a Phase 1/2 Gene Therapy Trial for Leber Congenital Amaurosis (LCA)

Shalesh Kaushal,1 Margaret Humphries,1 Elena Filippova,1 George Asdourian,1 Radouil Tzekov,1 Michael Stalvey,2 Terrence Flotte.2

1 Ophthalmology, University of Massachusetts School of Medicine, Worcester, MA; 2 Pediatrics, University of Massachusetts School of Medicine, Worcester, MA.

A number of recent reports have demonstrated long-term improvements in photoreceptor function in patients with Leber Congenital Amaurosis (LCA) due to mutations of RPE65 after subretinal injection of recombinant adeno-associated virus serotype

2 (rAAV2-hRPE65) vectors Our group initiated a phase 1/2 study of one of these vectors (rAAV2-CB-hRPE65) in July 2009, anticipating dosing a total of 12 participants (4 groups of 3) Group 1 will consist of adults (18 yrs and older) receiving 1.8x1011vg in 450 µls Group 2 will include children 8 to 17 years old at that same dose Groups 3 and 4 will consist of adults and children, respectively, receiving 6.0x1011vg

these doses and volume represent a step forward to a clincially relevant dosing scheme Safety outcomes will include monitoring for both ocular and non-ocular toxicities and ef cacy measures including visual  elds, visual acuity and electroretinography In the  rst treated patient, safety assessment from the  rst 6 months after injection have indicated only mild transient adverse events, consisting of the sensation of bright  ashes of light and some conjunctival congestion

Ef cacy measures indicate an increase in visual  elds and subjective improvements in visual function Further enrollment and follow-on results are anticipated

445 Positive Responses in a Mouse Model of Leber Congenital Amaurosis after Treatment with Compacted DNA Nanoparticles

Kiichiro Okano,3 Tadao Maeda,1,3 Linas Padegimas,2 Tomasz H

Kowalczyk,2 Sharon M Oette,2 Christopher R Gedeon,2 Ozge Sesenoglu-Laird,2 Susannah L Hyatt,2 Karla A Dines,2 Elena Tyr,2

Nikia L Beal,2 Robert C Moen,2 Krzysztof Palczewski,3 Mark J

Cooper.2

1 Ophthalmology, Case Western Reserve University, Cleveland, OH;

2 Copernicus Therapeutics, Inc., Cleveland, OH; 3 Pharmacology, Case Western Reserve University, Cleveland, OH.

Purpose: Compacted-DNA nanoparticles are a highly-ef cient and

non-toxic non-viral gene delivery system Human vision is initiated by photon absorption in visual pigments of retinal photoreceptors in the

eye Continuous regeneration of visual pigment 11-cis-retinal depends

on an enzymatic pathway in photoreceptor and retinal pigmented epithelial (RPE) cells known as the visual cycle, which is essential to maintain human vision Dysfunction of the visual cycle due to DNA mutation causes various inherited-retinal degenerative diseases, such

as Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP)

The purpose of this study is to evaluate the ef ciency and speci city of gene delivery by the nanoparticles in the wild-type (WT) mouse retina and the restoration of visual function in an LCA mouse model, the

lecithin:retinol acyl transferase (LRAT) gene-de cient mouse (Lrat -/-),

which is lacking 11-cis-retinal and other retinoid intermediates in the

eye Methods: To evaluate duration of functional gene expression

by nanoparticle-mediated gene delivery, nanoparticles containing the luciferase gene controlled by the polyubiquitin C promoter were injected into the subretinal space of WT mice The enzymatic activity of luciferase was visualized and quanti ed by Bioluminescent Imaging (BLI) Luciferase activity in mouse eye lysates was evaluated

by a biochemical assay To examine gene delivery to speci c target cells in the mouse retina, nanoparticles containing the enhanced yellow  uorescent protein (eYFP) gene, transcriptionally controlled

by the RPE cell-speci c human VMD2 promoter (hVMD2), were

injected into the subretinal space of WT mice eYFP expression was evaluated by spectral analyses and immunohistochemistry by confocal microscopy To evaluate therapeutic ef cacy for retinal

degeneration, nanoparticles with the Lrat gene controlled by the hVMD2 promoter were injected into the subretinal space of Lrat -/- mice and functional recovery was evaluated Results: Maintained

and high level luciferase activity was observed by BLI from days 2

to 200 after injection in the dosed eyes At day 277, luciferase lysate activity was over 3 logs above background eYFP emission and protein expression were detected speci cally in the RPE cell layer at day 21 after injection in the dosed eyes Improvement of electroretinogram

responses was observed in Lrat -/- mouse eyes treated with Lrat DNA

nanoparticles compared to those dosed with saline Signi cant levels

of retinoids and immunohistochemical evidence of LRAT expression

were detected in treated eyes as well Conclusion: Compacted-DNA

nanoparticles can ef ciently deliver genes to the mouse retina and produce long-term and tissue-speci c gene expression These data illustrate the therapeutic potential of the nanoparticles to treat various human retinal degenerative diseases

446 Adeno-Associated Virus Mediated Gene Therapy with a Rhodopsin Ribozyme for Treatment

of Autosomal Dominant Retinitis Pigmentosa (Adrp)

Haoyu Mao,1 Marina S Gorbatyuk,1 William W Hauswirth,1,2

Alfred S Lewin.1

1 Department of Molecular Genetics and Microbiology, University

of Florida, Gainesville, FL; 2 Department of Ophthalmology, University of Florida, Gainesville, FL.

Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations in the RHO gene, which codes for the opsin

of rod photoreceptor cells We are engaged in development of gene therapies for ADRP using adeno-associated virus (AAV), which ef ciently infects and transduces photoreceptor cells In the present set

of studies, we designed and constructed a ribozyme (Rz1) which can ef ciently cut human rhodopsin (RHO) gene but not the “hardened”

form of RHO The “hardened” form of RHO is a silent mutant RHO which speci cally resistant to degradation by Rz1 but expressing

a normal RHO HEK 293 cells were co-transfected with a plasmid carrying RHO cDNA and a chemically synthesized Rz1 Reduction

of RHO mRNA was detected by alkaline phosphastase activity in vitro Rz1 suppressed the RHO with 95% reduction of phosphatase activity compared with that of a control ribozyme We are testing gene therapy in a mouse model of ADRP that expresses a human transgene with a prevalent ADRP mutation: proline 23 substituted by histidine (P23H) Following AAV delivery of Rz1 to P23H transgenic mice in

a background of C57BL/6 wild type mice, the retinal degeneration

of injected eyes was monitored by the electroretinography (ERG), a non-invasive technique that measures the activity of photoreceptors and retinal inter neurons AAV delivery of Rz1 increased maximum a- and b-waves ERG responses, relative to eyes injected with a control virus These results encourage us to test the replacement of normal rhodopsin mRNA by the construction of an AAV vector expressing both Rz1 and ribozyme-resistant RHO (RHORz1) combination

447 POD DNA Nanoparticles Rescue an Adult Mouse Model of Retinal Degeneration

Sarah P Read, Siobhan M Cashman, Rajendra Kumar-Singh

Ophthalmology, Tufts University, Boston, MA.

Purpose: POD, or “Peptide for Ocular Delivery,” is a novel synthetic cationic cell penetrating peptide (CPP) which when modi ed

by addition of a polyethylene glycol moiety (PEG-POD) can be used

to compact plasmid DNA into 120-150 nm nanoparticles These nanoparticles can transfect the retinal pigment epithelium (RPE)

following delivery to the subretinal space of adult mice To test the physiological relevance of PEG-POD mediated gene delivery, PEG-POD DNA nanoparticles carrying a transgene expression cassette coding for Glial-cell line Derived Neurotrophic Factor (PEG-POD∼GDNF) were examined for their ability to rescue photoreceptors

in a light induced model of retinal degeneration Methods: Four days following subretinal delivery of PEG-POD∼GDNF, PEG-POD∼Lux (nanoparticles expressing a luciferase transgene) or buffer alone, retinal degeneration was induced by exposure of mice to bright blue light for 4 hours Expression of GDNF was con rmed using RT-PCR Apoptosis was quanti ed by both TUNEL and nucleosome release ELISA Anatomical and functional rescue was measured at 7 and 14 days post-light exposure by quanti cation of the outer nuclear layer (ONL) thickness and by electroretinogram (ERG), respectively Results: Retinas injected with PEG-POD∼GDNF showed a signi cant (∼7-fold) decrease in the number of TUNEL positive cells in the ONL relative to those injected with either PEG-POD∼Lux (p<0.005) or buffer (p<0.0005) Nucleosome release assay con rmed this reduction

in apoptosis (∼3.1-fold) in PEG-POD∼GDNF injected eyes relative

to both controls (p<0.05) The average ONL thickness of the superior retina in PEG- POD∼GDNF injected eyes was ∼31.5% greater at 7 days post-light treatment relative to PEG-POD∼Lux (p<0.01) or buffer alone (p<0.0005) and ∼26% greater than either control injection

at 14 days post-light treatment (p<0.05) When examining the functional response of the retina to light by ERG, PEG-POD∼GDNF injected eyes exhibited a 32-39% increase in amplitude in the a-wave (p<0.05) and a 27-31% increase in the b-wave amplitude (p<0.05) relative to controls after light treatment Conclusions: This study demonstrates histological and functional rescue of an adult model of retinal degeneration using a non-viral approach Though expression

is short lived for application in the clinic, this study represents an important step in the development of non-viral gene therapy for retinal diseases

448 Protection Against Laser-Induced Choroidal Neovascularization Following Delivery

of an Adenovirus Expressing a Soluble Form of Human CD59 to Murine Retina

Siobhan M Cashman,1 Kasmir Ramo,1 Rajendra Kumar-Singh.1

1 Ophthalmology, Tufts University, Boston, MA.

Purpose: Polymorphisms in a number of complement genes have

been shown to be either strongly predictive of or protective against Age-related Macular Degeneration (AMD) In patients with AMD, deposition of the Membrane Attack Complex (MAC) on the retinal pigment epithelium (RPE), Bruch’s membrane, and choroidal blood vessels has been observed Choroidal neovascularization (CNV) in the classic laser-induced mouse model of wet AMD has been shown

to be dependent on the formation of MAC We have previously shown that in vivo delivery of an adenovirus vector expressing human membrane-associated CD59 protects against human MAC deposition

on murine ocular tissues, including the RPE Protection against the effects of MAC was limited to those cells expressing the membrane associated CD59 We now wished to test the hypothesis that delivery

of an adenovirus vector expressing a soluble form of human CD59 (sCD59) can block the formation of laser-induced CNV, and can do so also when vector is administered distal from the site of laser burn

Methods: Murine hepatocytes (Hepa1c1c7) cells were incubated

in 1% normal human serum (NHS) diluted in media collected from human ARPE-19 cells infected with either an adenovirus expressing sCD59 or GFP (AdCAGsCD59, AdCAGGFP) The resulting lysis was measured by  uorescence activated cell sorting (FACS) analysis

of propidium iodide uptake Either an AdCAGsCD59 or a control virus (AdCAGpA) was injected into the subretinal space of C57Bl6/J mice, and 72h post-injection the mice were subjected to argon laser

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