536 long lasting vision following one time gene therapy: partnership of brain and retina revealed by fMRI

536 Long Lasting Vision Following One Time Gene Therapy Partnership of Brain and Retina Revealed By fMRI Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene &[.]

Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy

S208

SENSORY (OPHTHALMIC AND AUDITORY) DISEASES

534 Evaluating Competition of Delivered Small

Hairpin RNAs with Endogenous microRNAs for

Safe Effective Gene Knockdown

Paul N Valdmanis,1 Shuo Gu,2 Leszek Lisowski,1 Lan Jin,2 Kirk

Chu,1 Feijie Zhang,1 Yong Huang,1 Mark A Kay.1

1 Pedatrics and Genetics, Stanford University, Stanford, CA;

2 Center for Cancer Research, National Cancer Institute, Frederick,

MD.

RNA interference (RNAi)-based therapeutics have the potential to

revolutionize the way medicine, in particular personalized medicine,

is provided and delivered Knockdown of a disease gene by small

hairpin RNA (shRNA) delivery involves a delicate ratio between

effective concentrations of the shRNA within the cell and enabling

sustained normal activities within the cellular milieu Too much

expressed shRNAs can lead to toxicity and lethality in mice; thus an

evaluation the toxicity of existing delivery mechanisms is relevant in

this context such that this toxicity can be recognized and avoided We

have harnessed the potential of high throughput sequencing to identify

an endogenous signature of shRNA toxicity in the liver following

adeno-associated viral vector delivery and are working to establish the

downstream effects that result from this toxicity Mice were monitored

for elevated serum enzymes indicative of toxicity and the livers of

these mice were subjected to high throughput sequencing of small

RNAs including microRNAs As the level of small RNAs associated

with our delivered vectors increased, we noted a concomitant decrease

in the liver-specifi c microRNA, miR-122, while all other microRNAs

were unaffected in terms of relative expression in the liver Upon

evaluation of length differences of mature miR-122-5p we identifi ed

a specifi c loss of the 22 nucleotide isoform of miR-122 in toxic liver

samples RNA sequencing of the same livers indicates that predicted

and validated targets of miR-122 are de-repressed in affected livers

leading to displaced metabolic pathways, in particular related to

glucose and cholesterol metabolism We have validated some of

these miR-122 targets and are evaluating how the perturbation of

these genes relates to the cell death and toxicity we observe By

understanding the endogenous mechanisms that are compromised in

shRNA delivery, we can ensure that they are monitored and sustain

their expression to ensure safe and effective RNAi gene therapy

535 In Vivo Study of a Bi-Functional

Short-Hairpin RNA (bi-shRNA) Targeting Multiple

Single-Nucleotide KRAS Mutations in Pancreatic Cancer

Donald D Rao,1,3 Chris M Jay,1,3 Zhaohui Wang,1,3 Padmasini

Kumar,1,3 Neil Senzer,1,2,3 John Nemunaitis.1,2,3

1 Gradalis, Inc., Dallas, TX; 2 Mary Crowley Cancer Research

Centers, Dallas, TX; 3 Strike Bio, Inc., Dallas, TX.

KRAS is a member of guanine nucleotide-binding protein family

and acts as a key molecular switch regulating multiple intracellular

signaling pathways including epidermal growth factor receptor

(EGFR) KRAS mutations essentially result in constitutively active

KRAS and unregulated downstream signaling The majority of

pancreatic ductal adenocarcinoma (PDAC) patients carry a

single-nucleotide KRAS mutation, >90% of which are single amino acid

substitutions of Glycine at codon 12 in exon 2 The fi ve-year survival

rate of PDAC is less than 5% KRAS mutation-specifi c targeting

remains elusive and recent small molecule therapeutics demonstrates

weak affi nity or rapid degradation We have recently developed a

novel bi-functional shRNA (bi-shRNA) RNA interference (RNAi)

technology that achieves in vitro selective knockdown of mutant

KRAS expression (Figure 1).

We systematically determined the relative normal/mutant allele discriminating knockdown activity of a series of constructs by sequential evaluation of guide strand position effect The single nucleotide mutation at positions 2-5 of the guide strand to be the most effective Similar constructs were made and tested for G12D,

G12V, G12R (comprising 91% of PDAC KRAS mutations) and G12C

mutations (2.9%) G12D and G12C selective bi-shRNA expression

vectors did not inhibit HEK-293 (KRASwt) growth in contrast to cell

growth reduction of PANC-1 (KRASwt/G12D) and MiaPaCa2 (KRASwt/

multiplex knockdown targeting G12D, G12C and G12R were

determined to be effective for multiple expressive KRAS mutant

alleles with a single vector construct A second multiplex construct targeting G12C, G12D and G12V achieved similar results Mice are a biorelevant species for the triple bi-shRNA KRAS lipoplex

Preliminary safety testing revealed no toxic effect in vivo following

a single intravenous dose of liposome encased bi-shRNA KRAS triplexes In vivo effi cacy studies with PANC-1 or MIAPaCa-2

pancreatic cancer models will be reported Application of this therapeutic formulation with combination to EGFR targeting agents will also be examined

Sensory (Ophthalmic and Auditory) Diseases

536 Long-Lasting Vision Following One-Time Gene Therapy: Partnership of Brain and Retina Revealed By fMRI

Manzar Ashtari,1 Amanda Viands,1 Kathleen Marshall,1 Kenneth Shindler,2 Albert Maguire,2 Jean Bennett.2

1 Children’s Hospital of Philadelphia, Philadelphia; 2 University of Pennsylvania, Philadelphia.

Background:Human LCA2 patients treated with subretinal gene

therapy (GT) have shown dramatically improved and stable vision for a long time These improvements have been mostly attributed to the rescued retinal cells, but vision develops from combined work between the eye and brain and the role that the brain plays in the success of GT is not well studied Utilizing fMRI, we previously showed that GT enhances visual cortex responsiveness to visual inputs In this report, we examine the long-term effects of GT on plasticity of the visual pathway and the role it may play in augmenting vision

Methods: Follow-up neuroimaging studies were performed in six

LCA2 patients who underwent a second GT in their untreated eye Subjects received fMRI before the second eye GT (baseline), 1,3, and

6 months as well as 1,2, and 3 years after re-administration Visual stimuli consisted of high contrast checkerboard, and to account for variability in disease fMRI data was analyzed individually

Results: None of the subjects showed signifi cant activation within

the primary visual cortex (V1) on their baseline fMRI, except one young participant One month after GT, results showed dramatic increases in V1 activation that continuously improved over time Consistent with their cortical activation patterns, subjects showed no

Molecular Therapy Volume 22, Supplement 1, May 2014

Copyright © The American Society of Gene & Cell Therapy S209

SENSORY (OPHTHALMIC AND AUDITORY) DISEASES

LGN activation at baseline At one month follow up, LGN activation

unexpectedly initially appeared only on the side of the previously

treated eye, and by the end of two years fMRI follow up results

showed the expected bilateral LGN activation, as normally observed

in sighted controls fMRI results for all LCA2 patients showed a

similar pattern of activations However, younger subjects showed

even greater improvement over time than older participants

Conclusions: The eye and brain work in concert to enable vision

Using fMRI, we demonstrate the vital role that the brain plays in

securing long-lasting vision after one-time GT Visual improvement

was detected one month after GT and continually enhanced 2-3

years later LGN responses suggest that brain at fi rst uses previously

strengthened pathways promoted by the GT of the fi rst eye Over

time, brain plasticity was detected by observing increased activations

for ipsilateral LGN and bilateral V1 In summary, fMRI results

demonstrate that while GT initiates the sight in LCA2 patients, their

continual visual experiences promote long-term neural plasticity,

which in turn further enhances their vision Results suggest one-time

subretinal injection promotes continuous brain plasticity, leading to

progressive enhancement of visual pathway function and long-term

visual improvement Longer follow up studies are needed to further

support this hypothesis

537 Gene Therapy for X-Linked Retinitis

Pigmentosa: A Long-Term Effi cacy Study in a

Mouse Model of RPGR Defi ciency

Zhijian Wu,1 Suja Hiriyanna,1 Haohua Qian,1 Suddhasil

Mookherjee,1 Kayleigh Kaneshiro,1 Maria Campos,1 Chun Gao,1

Robert Fariss,1 Tiansen Li,1 Peter Colosi,1 Anand Swaroop.1

1 National Eye Institute, National Institutes of Health, Bethesda,

MD.

Retinitis pigmentosa (RP) refers to a diverse group of hereditary

retinal dystrophies characterized by a progressive loss of photoreceptor

cells, resulting in vision loss or blindness The prevalence of the

disease is approximately 1/3500 worldwide X-linked RP (XLRP)

accounts for 15% of all RP cases and is one of the more severe forms

of retinal degeneration Over 70% of XLRP cases are caused by

mutations on the Retinitis Pigmentosa GTPase Regulator (RPGR)

gene, one of the most common causes of RP among the over sixty

RP-causing genes identifi ed to date To develop a gene replacement

therapy for this disease, we designed and generated AAV vectors

carrying mouse or human RPGRorf15 full-length cDNA, and

conducted a long-term effi cacy study in an Rpgr knock-out (KO)

mouse model This model recapitulates the major features of the

human disease over a 20 month frame AAV8 or AAV9 vectors were

injected subretinally into 6 to 8 week- or 12 month-old Rpgr-KO mice

at doses ranging between 1e8 and 1e10 vector genomes (vg) per eye

The mice were monitored for retinal function and structural changes

by electroretinography (ERG) and optical coherence tomography

(OCT) and were sacrifi ced at 20-24 months for histological and

immunohistochemical analyses Our results showed that preservation

of retinal function and structure in Rpgr-KO mice was achieved with

either mouse or human RPGRorf15 vector administration Among

all tested doses, 3e8 vg or 1e9 vg per eye appeared to be optimal

for the mouse or human RPGRorf15 vector injection, respectively

The mice receiving these vector doses displayed signifi cantly larger

scotopic and photopic ERG amplitudes in their vector-injected eyes

compared to fellow control eyes, at 18 months post-injection OCT

analyses revealed the preservation of outer nuclear layer (ONL) in the

vector-injected retina, which was later confi rmed by histology Cone

opsin mislocalization was partially corrected as well Interestingly, the

mice injected at 1 year of age also displayed larger ERG amplitude

and a greater ONL thickness The mice receiving other doses of the

vectors generally exhibited suboptimal therapeutic effects, however

very high doses, such as 1e10 vg of the mouse or human RPGRorf15

vector, produced pronounced retinal toxicity We conclude that both mouse and human RPGRorf15 AAV vectors are able to prevent photoreceptor degeneration and preserve rod and cone function in the

Rpgr-KO mouse retina Our studies also suggest that patients with

RPGR mutations could benefi t even when treated at an advanced age

538 Gene Therapy With the Mitochondrial Heat Shock Protein 70 Suppresses Axonal Degeneration and Vision Loss in Experimental Optic Neuritis

Venu Talla,1 Vittorio Porciatti,1 Vince Chiodo,2 Sanford L Boye,2

William W Hauswirth,2 John Guy.1

1 Ophthalmology, Bascom Palmer Eye Institute, Miami, FL;

2 Department of Ophthalmology, University of Florida, Gainsville, FL.

PURPOSE: To rescue visual loss and optic neuropathy in the experimental autoimmune encephalomyelitis (EAE) mouse model using gene therapy with the mtHSP70 chaperone responsible for import of proteins into the mitochondria

METHODS: EAE was induced in DBA/1J (n=20) mice by subdermal injection of 0.1 ml homologous spinal cord emulsion in complete Freunds adjuvant Ten mice were rescued by intravitreal injection of ssAAV-CBA-mtHSP70 with a Flag tag, 10 were injected with the scAAV-Cox8-mCherry as injection controls Unsensitized DBA1J mice (n=10) were injected with scAAV-Cox8-mCherry as a control Visual function was assessed by pattern electroretinograms (PERG) Spectral domain OCT evaluated the thickness of the inner plexiform layer + nerve fi ber layers at 1, 3 and 6 months post injection (MPI) All mice were euthanized at 6MPI Retinas and optic nerves (ONs) were dissected for histological and ultrastructural evaluation Mitochondrial complexes I+III, III and I activities were analyzed in the retinas obtained from 4 MPI control, mCherry and EAE-mtHSP70 rescued mice Expression of EAE-mtHSP70Flag in the retina and ONs was evaluated at 1 MPI by immunofl uorescence (IF), RT-PCR and western blotting (WB) Mitochondrial import of Cox8-mCherry in retina of EAE mice with and without mtHSP70 rescue was analyzed RESULTS: IF revealed a typical punctate and perinuclear expression of Flag-HSP70 which colocalized with porin and thy1.2 RT-PCR, WB confi rmed HSP70 expression in the retina and ONs PERG analysis at 3 and 6MPI showed a 42% and 45% reduction

in amplitude in EAE-mCherry compared to control mCherry mice (p<0.005,) MtHSP70 rescued mice also showed a signifi cantly (p<0.05) reduced amplitudes (26% and 27% respectively) relative

to non EAE mice, however relative to EAE mice treatment rescued the amplitude by 37% and 41% respectively (p>0.05) PERG latency was delayed by 15% and 21% in EAE-mCherry compared to mCherry control (p<0.05), whereas the mtHSP70 treated mice rescued the delay by 100% and 83% respectively at 3 and 6MPI OCT images showed a signifi cant thinning in EAE-mCherry compared to mCherry control at 3MPI (16%) and 6MPI (15%) p<0.05, whereas mtHSP70 treatment rescued this thinning by 90% and 100% respectively (p<0.05) Ultrastructural analysis and histology of EAE ONs and retina indicated signifi cant loss of axons (41% loss vs control, p<0.01) and retinal ganglion cells (RGCs) (40% loss vs control, p<0.01) whereas, overexpression of mtHSP70 signifi cantly rescued axonal (85%, p<0.01) and RGC (85%, p<0.001) numbers Complex I+III, III and I activities in EAE mouse retinas decreased by 34%, 40% and 28% respectively vs control (p<0.05) and overexpression of mtHSP70 rescued these activities to near normal Overexpression of mtHSP70 improved mitochondrial protein import

CONCLUSIONS: AAV mediated mtHSP70 gene therapy preserves vision function and prevents degeneration of the ON that causes permanent disability in optic neuritis and MS patients unaltered by commonly used disease modifying drugs that target infl ammation