366 development of quantitative taqman rt pcr for the evaluation of non viral mediated gene transfer to the airways

Cystic fibrosis CF is considered a disease that could be treated by gene therapy, yet the results from past clinical trials showed only transient transgene expression.. In this study, we

Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy

S142

GENE THERAPY APPROACHES TO PULMONARY DISEASE

Model for Cystic Fibrosis Gene Therapy

Haeyul Lee,1,2 David R Koehler,1 Cho Y Pang,1,3 Ronald H

Levine,3 Philip Ng,4 Donna J Palmer,4 Paul M Quinton,5 Jim

Hu.1,2

1 Research Institute, The Hospital for Sick Children, Toronto, ON,

Canada; 2 Institute of Medical Science, Univeristy of Toronto,

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

Toronto, Toronto, ON, Canada; 4 Department of Molecular and

Human Genetics, Baylor College of Medicine, Houston, TX;

5 Department of Pediatrics, University of California, San Diego,

La Jolla, CA.

Cystic fibrosis (CF) is considered a disease that could be treated

by gene therapy, yet the results from past clinical trials showed

only transient transgene expression Gene therapy vectors are mostly

studied in cultured cells, rodent models or non-human primates, but

it is difficult to test them in human system prior to clinical studies

In this study, we investigated the possibility of using human sweat

glands as a model for testing CF gene therapy vectors In order to

deliver genes to human sweat glands ex vivo, we explored various

gene delivery methods using a helper-dependent adenovirus vector

containing cytokeratin 18 regulatory elements Gene delivery to

sweat glands in skin organ culture by topical application,

intra-dermal injection or submerged culture was not effective We were

able to enhance transduction efficiency by isolating and pre-treating

sweat glands with dispase, which indicates that the basement

membrane is a critical barrier to gene delivery by adenoviral vectors

Using this approach, we showed that the Cftr gene could be efficiently

delivered to and expressed by the epithelial cells of sweat glands

with our helper dependent adenoviral vector Based on our study

we propose that sweat glands can be used as an alternative model to

study functional efficacy of CF gene therapy in humans

Recombinant Adenovirus-Mediated Transfer of

Soluble TGF- βββββtype II Receptor Gene

Koichi Takayama,1 Haiping Zhang,1,2 Junji Uchino,1 Akiko

Harada,1 Taishi Harada,1 Yoichi Nakanishi.1

1 Research Institute for Diseases of the Chest, Kyushu University,

Fukuoka, Japan; 2 Department of Oncology, Shanghai

Pulmonology Hospital, Shanghai, China.

Radiotherapy is an important conventional therapeutic modality

in the treatment of the malignant thoracic diseases, such as lung

cancer However, the radiation-induced injury to the normal lung

tissue, including radiation pneumonitis and lung fibrosis, is sometimes

severe and even lethal, and thus, becomes the main dose limiting

factor TGF-β and its receptor are known to play a pivotal role for

the radiation induced lung damage In this study, we confirmed that

following thoracic irradiation with a single dose of 9 Gy,

radiation-induced TGF-β1 concentration elevated in serum with biphasic

pattern Therefore, we constructed a replication-defective recombinant adenovirus carrying the soluble TGF-β type II receptor cDNA (AdsTGFβRc), which can infect the cells and produce the soluble form of TGF-β type II receptor This soluble receptor can bind TGF-β in a dominant-negative form, and consequently block the TGF-β function In vivo experiment, i.p injection of the AdsTGFβRc in mice before irradiation could suppress the TGF-β1 serum elevation and reduced the pneumonitis histologically

Helper-Dependent Adenovirus Formulation to Rabbit Lung Using an Intratracheal Catheter

David R Koehler,1 Helena Frndova,1 Kitty Leung,1 Emily Louca,1

Donna Palmer,2 Philip Ng,2 Colin McKerlie,1 Peter Cox,1 Allan L Coates,1 Jim Hu.1

1 Program in Lung Biology Research, Hospital for Sick Children, Toronto, ON, Canada; 2 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.

Poor transduction of the ciliated airway epithelium is a common difficulty encountered in lung gene therapy trials with large animals and humans We delivered a helper-dependent adenovirus vector, incorporating a human epithelial cell-specific expression cassette,

to rabbit lung An intratracheal device was used to aerosolize a moderate dose of virus mixed with the enhancing agent LPC (lysophosphatidycholine), directly into the airways Lung mechanics, body weight and temperature, transgene expression and histopathology was studied at day 5 Transgene expression was seen in the epithelium of large and small airways, from trachea to terminal bronchioles, with a strong tendency toward the right lung All cell types of the surface epithelium were transduced Extensive transduction of the epithelium (66% of cells in trachea) was obtained using virus formulated in isotonic 0.1% LPC, while virus formulated

in 0.01% LPC transduced fewer cells (24% in trachea) Fever and a transient decrease in dynamic lung compliance was observed following aerosol delivery Mild-to-moderate patchy pneumonia without edema was also observed These data demonstrate a strategy for effective transduction of airway epithelium in a large animal

RT-PCR for the Evaluation of Non-Viral Mediated Gene Transfer to the Airways

Ian A Pringle,1,2 Rebecca L Smith,1,2 Bryony L Jones,1,2

Deborah R Gill,1,2 Stephen C Hyde.1,2

1 GeneMedicine Research Group, University of Oxford, Oxford, Oxfordshire, United Kingdom; 2 The UK Cystic Fibrosis Gene Therapy Consortium, United Kingdom.

Gene therapy for cystic fibrosis lung disease will likely require repeated delivery of gene transfer agents (GTA) to the terminally differentiated cells of the airway epithelium We are currently testing several non-viral GTAs for efficiency of gene transfer following aerosol delivery to the lungs of mice and sheep The most sensitive method available to quantify gene expression from plasmid vectors

is quantitative (TaqMan) reverse transcriptase (RT)-PCR, which has been widely used to quantify gene expression in pre-clinical and clinical samples TaqMan PCR can potentially detect a single copy

of the target sequence per reaction We designed TaqMan assays to quantify both vector-specific mRNA and endogenous mRNA of a normalising gene Specifically, assays were developed for plasmids containing the CMV promoter, or the human polyubiquitin C (UbC) promoter, shown to have improved duration of expression in the lung In addition, assays have been developed to detect endogenous

murine and ovine Cftr mRNA Assays were designed to be specific

for mRNA by placing one of the primer sequences across an exon splice site In order to determine the absolute copy number of mRNA

Molecular Therapy Volume 11, Supplement 1, May 2005

GENE REGULATION: GENE TARGETING AND RNA

in a given sample, RNA standard curves were produced DNA

templates for the assays were constructed and included the PCR

amplicon plus surrounding sequences totalling 160bp RNA

standards were generated incorporating RNase resistant

ribonucleotides into RNA transcripts, using the Ambion Competitor

Construction Kit The mimics were quantified using the Molecular

Probes RiboGreen assay and dilutions were produced containing 1

to 510 copies/µl 1µl of each dilution was used in paired RT reactions

with sequence specific primers and MultiScribe RT Quantitative

PCR was then carried out using an Applied Biosystems TaqMan

7700 Sequence Detector and standard curves generated for the four

assays When paired (multiplexed), the CMV and sheep Cftr assays

were linear between 100 and 106 starting copies per reaction

(R2=0.984 and 0.981) The UbC and mouse Cftr assays were paired

and the UbC assay was linear between 100 and 106 starting copies

(R2=0.987) while the mouse Cftr assay was linear between 1500

and 106 starting copies per reaction (R2=0.890) Control samples

lacking RT did not result in amplification, indicating that the standards

were free of template DNA These results show the high degree of

sensitivity and linearity that can be obtained with TaqMan

RT-PCR These assays will now be used to quantify absolute levels of

expression in our pre-clinical models to determine the optimal GTA

formulation for future clinical studies

Following Intrapleural Administration of an AAV5

Vector Encoding Human α αα αα1-Antitrypsin to

Non-Human Primates

Ben-Gary Harvey,1 Bishnu P De,1 John P Ayala,1 Neil R

Hackett,1 Adriana Heguy,1 Ronald Crystal.1

1 Genetic Medicine, Weill Medical College of Cornell University,

New York, NY.

α 1-antitrypsin (α1AT), a 52 kDa serine proteinase inhibitor

secreted by the liver, provides >95% of the functional anti-protease

protection of the lower respiratory tract α1AT deficiency is an

autosomal recessive disorder associated with early-onset emphysema

if serum α1AT levels are <11 M (570 mg/ml) We have developed an

efficient strategy for gene therapy for the pulmonary manifestations

of α1AT deficiency based on intrapleural administration of AAV

vectors encoding human α1AT, thus providing local production of

α1AT at the target organ, resulting in persistent α1AT expression

at therapeutic levels We have previously demonstrated (De et al,

Mol Ther 2004;10:1003) that intrapleural administration of 1011

genome copies (gc) of an adenoassociated virus serotype 5 (AAV5)

cytomegalovirus-chicken β-actin hybrid promoter (AAV5CUhα1AT)

to C57Bl/6 mice resulted in α1AT serum levels of 900±50 µg/ml at

40 wk, >1.6-fold the accepted therapeutic dose in humans

Importantly, the α1AT level in the bronchoalveolar lavage fluid

(normalized to total protein) resulting from delivery of the

AAV5CUhα1AT to the pleura was comparable to that found in the

serum In preparation for a clinical trial, the present study addresses

the safety of administration of AAV5CUhα1AT to the pleural space

of non-human primates at doses scalable to humans (evaluation of

human α1AT levels in these animals is not technically feasible due

to the 94% homology between African green monkey and human

α1AT) The AAV5CUhα1AT vector (1013 gc/animal, n=3) and control

(AAV5-luciferase, 1013 gc, n=1) were administered under fluoroscopy

guidance in 5 ml of PBS into the right pleural space of juvenile

African green monkeys, using an 22 gauge angiocatheter in the 5th or

6th intercostal space Chest X-rays carried out immediately after

vector administration, and at days 3,14 and 56 post-vector

administration demonstrated the absence of pneumothorax, pleural

effusion, pneumonia or any other pulmonary complications Serum

was collected at 3 time points (days -30, -15 and 0) prior to vector

administration, to obtain baseline blood count and chemistry values, and at days 3, 14, 28, 56 and 91 post-vector administration There were no significant differences (p>0.05 all parameters) between baseline (pre-vector administration) blood counts at any time point, except for transient increases in white blood cell count at days 3 and

14 post-vector administration (p= 0.007), likely reflecting a physiological response to the procedure and/or vector administration There were no significant differences in blood chemistry values at any time point, with the exception of serum Ca++ levels that decreased from initially high values into the range for adult African green monkeys The anti-AAV5 neutralizing antibody titers post-vector administration ranged from 250 to 640 above background Together with the long term gene expression following gene transfer to mice, these findings support the concept of a clinical trials to assess the safety of intrapleural administration of AAV5CUhα1AT to individuals with α1AT deficiency

GENE REGULATION: GENE TARGETING AND RNA

CML-Derived CD34+ Cells to Inhibition of SHP2, Gab2 and Stat5 Gene Expression by RNA Interference (RNAi)

Michaela Scherr,1 Karin Battmer,1 Anuhar Chaturvedi,1 Beate Schultheis,2 Arnold Ganser,1 Matthias Eder.1

1 Hematology and Oncology, Hannover Medical School, Hannover, Germany; 2 III Med Clinic, University Hospital of Mannheim, University of Heidelberg, Mannheim, Germany.

RNA interference has rapidly become an efficient tool for functional genomics in a variety of organisms Stable expression of shRNA driven by pol III promoters upon lentiviral gene transfer can induce long-term gene silencing in mammalian cells We recently demonstrated that lentivirus-mediated anti bcr-abl RNAi can specifically silence bcr-abl gene expression, inhibit oncogene driven cell proliferation, and eradicate leukemic cells depending on the dose

of lentivirus-mediated shRNA expression However, since effective depletion requires a threshold of lentiviral integrations into target cell genomes, the risk of insertional mutagenesis may limit the therapeutic value of this approach To search for new potential therapeutic targets in CML, we studied the function of several signaling molecules in purified normal and CML CD34+ cells from chronic phase CML patients harvested at initial diagnosis We selected SHP2, Gab2, and Stat5 based on their constitutive tyrosine phosphorylation in bcr-abl + cell lines Several shRNAs for each target gene were evaluated using a bicistronic EGFP-encoding reporter plasmid system as described earlier Effective shRNA expression cassettes were cloned into lentiviral plasmids encoding RFP to track lentiviral transduction Lentivirus-mediated RNAi targeting SHP2, Gab2, or Stat5 results in a reduction of mRNA and protein by more than 90 % and induces a rapid depletion of transduced bcr-abl + and negative cell lines In addition, RNAi against all three targets enhances imatinib induced depletion of bcr-abl+ K562 cells We next transduced normal and CML-derived primary CD34+ cells with control and anti-SHP2, Gab2, and Stat5 lentiviruses, and analysed colony-formation of transduced, i.e RFP+ progenitor cells in methylcellulose cultures To eliminate effects of different transduction rates we plated CD34+ cells for each transfection in the presence of high (GM-CSF: 20 ng/ml; IL-3: 10 ng/ml) or low (GM-CSF: 0.2 ng/ml; IL-3: 0.1 ng/ml) cytokine concentrations indicating the functional relevance of the respective RNAi-target for CFU-colony formation Whereas anti-SHP2, Gab2, and Stat5 RNAi did not reduce the proliferation of normal transduced CFU (n=5), proliferation of transduced CFU from CML patients was specifically reduced between 50 to 85 % under low cytokine