122 efficient and sustained gene transfer to neuroepithelial and neuronal cells following intratechal injection of HDAd vectors

122 Efficient and Sustained Gene Transfer to Neuroepithelial and Neuronal Cells Following Intratechal Injection of HDAd Vectors Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The Amer[.]

Molecular Therapy Volume 17, Supplement 1, May 2009

ADENOVIRUS VECTOR BIOLOGY

pIX-mRFP1 and pV-EGFP by simultaneous detection allowed us

to observe the complete cycle of viral replication in infected cells

These results indicate that the simultaneous detection of adenovirus

using fl uorescent proteins is suitable for real-time analysis, including

identifi cation of infected cells, and monitoring viral spread, which will

be required for evaluation of oncolytic adenoviruses In conclusion,

the dual-labeling of adenovirus using two kinds of fl uorescent proteins

offers a unique way to more thoroughly monitor adenovirus infection

and to study adenovirus biology

Bone Regeneration by Thymidine Kinase and

Ganciclovir Treatments

Barbara Lombardo,1,2 Teresa Rocco,1 Maria Teresa Esposito,3

Bruno Cantilena,3 Sara Gargiulo,1,4 Arturo Brunetti,1,4 Donatella

Montanaro,1 Giancarlo Troncone,1,5 Francesco Salvatore,1,2,3 Lucio

Pastore.1,2,3

1 CEINGE-Biotecnologie Avanzate, Naples, Italy; 2 Biochimica e

Biotecnologie Mediche, Università degli Studi di Napoli Federico

II, Naples, Italy; 3 SEMM, European School of Molecular Medicine,

Naples, Italy; 4 Scienze Biomorfologiche e Funzionali, Università

degli Studi di Napoli Federico II, Naples, Italy; 5 Anatomia

Patologica e Citopatologica, Università degli Studi di Napoli

Federico II, Naples, Italy.

Background: Bone morphogenetic proteins (BMPs) belong to the

TGF-Beta superfamily and are important regulators of key events in

the processes of bone formation Strategies aimed at BMPs expression

in modifi ed cells have been successful in stimulating osteoblastic

differentiation However, unregulated secretion of BMPs has been

implicated in bone overproduction in the expression site.We tested

the ability of fi rst generation adenoviral vector expressing BMP-4

(FG-AdBMP-4) to drive bone regeneration after i.m.administration

Our experimental approach is focused on the regulation of BMP-4

expression using a thymidine kinase (TK) expression system after

i.m virus injection and daily i.p administration of gancyclovir, in

order to regulate gene expression and to control bone regeneration

Methods: We used 3 experimental athymic nude mouse’s groups

The group 1 received a FG-AdBMP-4 concentration of 5×1010 pv/

mouse into the right femoral quadriceps and a FG-AdBMP-4/TK

concentration of 5×1010 vp/mouse into the left femoral quadriceps

The group 2 received a FG-AdBMP-4 concentration of 9×1010 vp/

mouse into the right femoral quadriceps and a FG-AdBMP-4 /TK

concentration of 9×1010 vp/mouse into the left femoral quadriceps

The group 3 was utilized as control group At day 0, virus was

administered by i.m route, while ganciclovir was administered i.p

(50 mg/Kg) daily On day 1, 7, 14, 21, 28 the total calcifi ed tissue

area at the site of injection were measured by DEXA Part of the

mice of group 2 received gancyclovir starting on day 14 after vector

administration On day 28 the mice were sacrifi ced and muscle

samples were collected for histological analysis such as Alizarin

Red S staining and TUNEL assay Results: In vivo infection using

vectors expressing also the TK system exhibited a great effi cacy to

regulate BMP-4 expression Evaluation by DEXA which included

several parameters such as bone mineral density (BMD), bone mineral

concentration (BMC), bone area (BA), and tissue area (TA), showed

a reduction of bone formation in the areas where FG-AdBMP-4/TK

had been administered; in particular mice treated at day 14 showed

a reduction of bone formation whereas mice treated from day 0

showed absence of bone formation At the last time point, all mice

were sacrifi ced and the muscle samples were collected for each mice

Histological analysis was demonstrated for each tissue which showed

the presence of bone formation in the right quadriceps whereas, the

left quadriceps showed bone degeneration Alizarin Red S staining

was performed to visualize the calcium deposits in mineralized bone

TUNEL Assay showed presence of apoptotic cells in necrotic zones

that corresponds to the site of BMP-4TK injection in mice treated with gancyclovir

Angiotensin 1-7 Attenuates Angiotensin II-Induced Cardiomyocyte Hypertrophy

Monica Flores-Muñoz, Andrew H Baker, Stuart A Nicklin

BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom.

The renin-angiotensin system (RAS), has multiple regulatory functions, as well as pathophysiological effects in cardiovascular disease (CVD) such as cell proliferation, cardiac hypertrophy, infl ammation and oxidative stress via the actions of angiotensin II (AngII) Angiotensin converting enzyme 2 (ACE2), a homologue

of ACE, whose expression is restricted to heart, testis and kidney, metabolizes AngII to Ang1-7, suggesting the presence of local tissue-specifi c RAS Recent studies have indicated that Ang1-7 antagonises AngII signalling via the receptor MAS, indicating that maybe Ang1-7 is a useful therapeutic for CVD We used exogenous peptide and a gene transfer approach to study Ang1-7 effects in cardiomyocyte hypertrophy We developed adenovirus vectors that over-express Ang1-7 using an expression cassette encoding a renin signal peptide (to ensure secretion), a mouse heavy chain IgG (to provide mass for effi cient production of the protein), a furin protease cleaveage domain (to release the peptide) and Ang1-7 Effi cient expression of the Ang1-7 fusion protein in RAdAng1-7 transduced cardiomyocytes was confi rmed by western blot To study each peptide in cardiac hypertrophy an in vitro model was established in rat H9c2 cardiomyocytes and primary rabbit cardiomyocytes H9c2 cells were stimulated with 100nM AngII for 4 days to induce cellular hypertrophy The length of AngII-stimulated cells was 149.7±15.6nm (unstimulated = 112.3±13.5nm; p<0.05) Addition of exogenous Ang1-7 to AngII-stimulated H9c2 cells blocked hypertrophy (Ang1-7 = 118.5±11nm; p<0.05) Next H9c2 cells were transduced with RAdAng1-7 at 3 different doses (300, 500 and 1000 plaque forming units (pfu)/cell) following stimulation with AngII At 500 and 1000pfu/cell RAdAng1-7 inhibited hypertrophy Next, AngII stimulated rabbit left ventricular cardiomyocytes were transduced with RAdAng1-7 at 50, 100 and 300 pfu/cell RAdAng1-7 blocked AngII-induced hypertrophy in primary cardiomyocytes Additionally, co-incubation of RAdAng1-7 transduced cells with the MAS receptor antagonist A779 blocked the antihypertrophic effect, suggesting Ang1-7’s effects were mediated by the receptor MAS To confi rm this effect was a result of peptide secretion from RAdAng1-7 we performed a conditioned media assay HeLa cells were transduced with 100pfu/cell of RAdAng1-7 and 48 hours later conditioned media was transferred to AngII-stimulated H9c2 cells Conditioned media from RAdAng1-7 transduced HeLa cells inhibited AngII-induced hypertrophy In summary, we demonstrate that Ang1-7 antagonizes the hypertrophic response of cardiomyocytes to AngII stimulation, suggesting this may be a useful therapeutic approach to target cardiac remodeling in cardiovascular disease

122 Effi cient and Sustained Gene Transfer

to Neuroepithelial and Neuronal Cells Following Intratechal Injection of HDAd Vectors

Pasquale Piccolo,1 Scott V Dindot,1 Donna J Palmer,1 Nathan C Grove,1 Brunetti-Pierri Nicola.1

1 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.

Helper-dependent adenoviral (HDAd) vectors are devoid of all viral genes and result in long term transgene expression in the absence of chronic toxicity Because of their ability to infect post-mitotic cells, including cells of the central nervous system (CNS), HDAd vectors

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S48

ADENOVIRUS VECTOR BIOLOGY

are particularly attractive for brain gene therapy However, the brain

is a complicated target for vector delivery aiming to correct a disease

with diffuse involvement because is a complex organ with discrete

and intricate interconnections between various types of cells We

show that HDAd vectors administered into the cerebrospinal (CSF)

fl uid via cisterna magna injection in mice result in minimal systemic

toxicity and extensive transduction of neuroependymal cells as

well as neuronal cells Vector-transduction of neuroepithelial cells

can potentially deliver their transgene products to the whole CNS

through the ventricular circulation making this approach attractive

particularly for lysosomal storage disorders Moreover, the ability to

transduce neuronal cells can potentially result in clinical benefi t for

other neurodegenerative disorders

on Replication Competent and Replication

Incompetent Adenovirus

Makoto Sato,1 Lily Wu.1

1 Molecular and Medical Pharmacology, David Geffen School of

Medicine at University of California Los Angeles, Los Angeles.

Recombinant adenoviral vectors are one of the most currently

used gene therapy vectors due to their large capacity, broad

spectrum of infectivity including dividing and non-dividing cells,

lower pathogenicity and ease of production to high titers To date,

we have developed more than thirty recombinant vectors and have

successfully propagated these vectors to high titer However, recently

we have faced issues of ineffi cient production of vectors, where

the titer of our vectors have hardly exceeded 1010 plaque forming

unit per milliliter Surprisingly, analysis of these low titer stocks

through electron microscopy and PCR revealed contamination by

adeno-associated virus (AAV) Flexible primer sets designed within

the conserved regions in rep and cap genes of AAV successfully

detected 1.5 kb products Of the purifi ed stock samples evaluated,

one showed contamination by adeno-associated virus serotype 2

In order to predict and avoid future contamination of AAV in our

adenoviral preparations, we decided to investigate the infl uences of

the contamination in adenovirus production and the effects of using

these contaminated stocks in experiments Briefl y, no difference was

seen in transgene expression between the vector preparations with

and without AAV contamination, however vector production was

less effi cient in the preparations with AAV The contamination issues

that we faced, drew our attention to the oncolytic adenovirus stocks

that we have developed Since AAV replication requires helper virus

functions i.e., E1, E2, E4 and VA RNAs, issues of AAV contamination

are relevant not only for recombinant the adenovirus in 293 cells but

also for the oncolytic adenovirus in replication competent cell lines

that can potentially act as a helper for AAV

To Treat Spinal Muscular Atrophy

Milagros Risco Quiroz,1,2 Michael A Kennedy,1,2 Rashmi

Kothary,1,3 Robin J Parks.1,2

1 Regenerative Medicine, Ottawa Health Research Institute,

Ottawa, ON, Canada; 2 Biochemistry, Microbiology and

Immunology, University of Ottawa, Ottawa, ON, Canada;

3 Cellular and Molecular Medicine, University of Ottawa, Ottawa,

ON, Canada.

Spinal muscular atrophy (SMA) is a fatal autosomal recessive

disorder caused by reduced levels of the survival motor neuron (SMN)

protein, which results in degeneration of motor neurons, leading

to muscle weakness, and ultimately death To provide an effective

therapy for SMA, SMN expression must be restored The goal of our

research is to develop a gene therapy strategy for the treatment of

SMA Replication-defective adenovirus vectors (Ad) are commonly

used as gene delivery vehicles We have designed a modifi ed Ad that expresses SMN We show that Ad-SMN can be effi ciently expressed

in tissue culture models of SMA, restoring SMN protein levels We also demonstrate that SMN localizes to nuclear structures, called gems, in Hela cells Our studies indicate that Ad-SMN may provide

an effective gene therapy strategy for treating SMA

Vector Reduces Immune Reactivity and Increases Transgene Expression after Administration to Rat Submandibular Glands

Changyu Zheng,1 Nikolay P Nikolov,1 Bruce J Baum.1

1 Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD.

Recently, we reported that the hybrid vector AdLTR2EF1 α-hEPO signifi cantly extended transgene expression in the absence

of integration (Zheng et al, Mol Ther, 2008) AdLTR2EF1α-hEPO

is an E1 and partial E3 (E3 14.5k and E3 14.7k) deleted first generation serotype 5 adenoviral vector (Ad 5) vector It contains

an 858 bp DNA fragment [251 bp envelope sequence plus 607 bp long terminal repeat (LTR)] from Moloney Murine Leukemia Virus upstream of the human elongation factor 1 alpha (EF1α) promoter and human erythropoietin (hEPO) cDNA, with another LTR sequence downstream of the polyadenylation signal Adenoviral E3 proteins are immunoregulatory and affect host inflammatory responses

To determine if an E1/E3 deleted hybrid Ad 5 vector can result in improved transgene expression after administration to submandibular glands (SGs), we constructed Ad∆E1/3LTR2EF1α-hEPO, a vector similar to AdLTR2EF1α-hEPO except for more extensive E3 deletion Results showed that transgene expression from Ad∆E1/3LTR2EF1 α-hEPO transduced rat SGs was ∼15-fold lower than that seen with AdLTR2EF1α-hEPO transduced rat SGs at 1010 particles/gland (20.4 ± 5.8 and 304.8 ± 231.6 mU/ml serum on day 7, respectively) However, all rats (n=5) transduced with Ad∆E1/3LTR2EF1α-hEPO at this dose had sustained elevated hEPO levels and hematocrits for 6 months (length

of experiment) compared to only ∼2 months for the AdLTR2EF1 α-hEPO transduced rats Quantitative PCR demonstrated that ∼0.1%

of each vector genome remained in the SGs at 6 months The ratio of vector genome present in extracted high molecular weight DNA:low molecular weight DNA was ∼1, consistent with a low integration frequency with both vectors We next compared the immune reactivity elicited by these two vectors The median titer of anti-Ad5 antibody present in rat serum at 6 months was signifi cantly higher (p< 0.003) following AdLTR2EF1α-hEPO (median:1:512; range: 1:256-1:512, n=5) transduction of SGs than that found in rats whose SGs were transduced with Ad∆E1/3LTR2EF1α-hEPO (median: 1:4; range: 1:2-1:128, n=5) Interestingly, there was no difference in serum anti-Ad5 antibody levels when both vectors were administered intramuscularly The levels of several cytokines (IL-1a, IL-1b, IFNg, IL-10, MCP1 and MIP1a) in aqueous SG extracts were dramatically increased on day

2 from the AdLTR2EF1α-hEPO transduced rats compared to those seen with the Ad∆E1/3LTR2EF1α-hEPO transduced rats There were

no differences in the serum levels of these cytokines between the two groups These data strongly suggest that the Ad∆E1/3LTR2EF1 α-hEPO vector elicits much weaker local immune responses in rat SGs than seen after AdLTR2EF1α-hEPO vector administration, and this reduced immune reactivity is associated with signifi cantly longer transgene expression