774 a functional model system to evaluate spliceosome mediated RNA trans spicing using a lacz target reporter mouse

774 A Functional Model System To Evaluate Spliceosome Mediated RNA Trans Spicing Using a LacZ Target Reporter Mouse Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������© ���������[.]

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

Copyright © The American Society of Gene Therapy

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CANCER TARGETED GENE THERAPY II

sequence) and transcriptional silence in air across our cell line panel

(up to 8 fold reduction against various HRE constructs) TNFα has

been placed under the transcriptional control of this construct and

in vitro studies are ongoing In addition, we have cloned luciferase

driven by our novel CA9-promoter in an adenoviral context and are

currently evaluating whether the aerobic silencing apparent from in

vitro studies translates to tumour specific expression when delivered

systemically in nude mice Further refinement of this vector through

the incorporation of SREs is anticipated to give robust radiation

responsiveness resulting in the generation of a novel, tightly regulated,

dual responsive vehicle for cancer gene therapy

Egr-1 Promoter Mediated by GM-CSF in Human

Gliomas

Francisco Martinez-F,1,2 Erika Gomez,1 Joanne T Douglas,3

David T Curiel,3 Andres A Gutierrez.1

1 Cell Therapy Unit, Centro Nacional de Rehabilitacion, Mexico,

DF, Mexico; 2 Department of Pharmacology, School of Medicine.

UNAM, Mexico, DF, Mexico; 3 Gene Therapy Center, University of

Alabama at Birmingham, Birmingham, AL, United States.

Introduction Suicide gene transfer mediated by adenoviral

vectors is key factor for development of new treatments of gene

therapy for human gliomas A crucial goal for this strategy is modulate

the transcriptional process on the target cells for external factors

such as cytokines, hormones or physical agents Based on this

concept, we are testing several inductors to drive and modulate the

transcriptional activity of the egr-1 promoter on the luciferase

reporter gene Mueller et al., describe expression of Granulocyte

Macrophage-Colony Stimulating Factor (GM-CSF) receptors in

human glioma biopsies and autoregulated expression of receptors

In other hand, Sakamoto et al describe egr1-promoter activity

induced by GM-CSF in hematopoietic cells, fibroblasts and synovial

cells Hereby, we test to GM-CSF and its value as inductor for gene

therapy in different glioma cell lines Material and methods.

Adenoviral constructs have been generated based on the AdEasy

system by homologous recombination in bacterial cells The –600

pb fragment of egr-1 Promoter was cloned upstream of the luciferase

gene into the MCS of pShuttle plasmid Adenoviral plasmid generated

was packaged into HEK293 cells, screened, purified for a large scale

production and titer for further experiments Glioma Cell lines

(CH235, D54 and U373) were cultured in standard conditions with

D-MEM media containing 10% of FBS and 1% of Antibiotics 1x

105cells were seeded and infected with 25 MOI for 2 hours in low

serum OPTIMEM media (Invitrogen) After that, cells were washed

and maintained in D-MEM media containing 1 % of FBS for 24 hrs

before induction with 1 nM and 3 nM GM-CSF Stimulated cells

with 20 % of FBS were used as positive control Proteins were

obtained using Cell culture lysis (Promega Corp.) and stored at –20°

C Luciferase Activity was quantified using Luciferase Assay System

(Promega Corp.) in a Victor Wallac instrument according to

manufacturer instructions All experiments were performed for

triplicate and luciferase activity was corrected to protein amount

Results and conclusions For CH235 cell line, maximum response

was reached at six hours after induction with 1 nm and 3 nM of

GM-CSF This response was similar or higher than positive control

and compared to basal expression means 75% of luciferase activity

In D54 cell line stimulated with 3 nM of GM-CSF shows 3 holds of

luciferase activity compared to basal control at 12 hrs U373 Cell

line shows 4.5 holds of luciferase activity at 3 nM of GM-CSF and

slightly upper than the positive control We are exploring the way

to enhance this response and realizing comparative studies with

other promoters and inductors

Gene Therapy

Wing-Shing Cheng,1 Valeria Giandomenico,1 Magnus Essand.1

1 Clinical Immunology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.

A promoter, driving the transcription of a tissue specific gene can

be used to restrict therapeutic gene expression in target cells from the same tissue Prostate cancer is a particularly appropriate target for such gene therapy approach since the prostate gland is non-essential for life and a number of prostate specific promoter and enhancer elements have been characterized

TARP (T cell receptor γ chain alternate reading frame protein) is

a protein that in males is uniquely expressed in epithelial cells within the acinar ducts of the prostate and in prostate cancer cells Here we describe the TARP promoter and its potential use in prostate cancer gene therapy We found that the proximal TARP promoter contains

a functional androgen response element and that TARP expression

is regulated by testosterone at the transcriptional level through androgen receptor activation Luciferase reporter gene assays revealed that the basal activity of the TARP promoter is higher than the activity of the PSA promoter Chimeric regulatory sequences containing the TARP promoter together with enhancers from prostate specific antigen (PSA) and/or prostate specific membrane antigen (PSMA) were constructed A regulatory sequence consisting of the TARP promoter and the PSA enhancer gives 20 times higher activity than a sequence consisting of the PSA promoter and PSA enhancer The TARP promoter/PSA enhancer regulatory sequence retains its prostate specificity, although its transcriptional activity is strictly controlled by testosterone A regulatory sequence consisting of the TARP promoter and the PSMA enhancer gives high prostate specific expression also under testosterone-depleted conditions Prostate cancer patients are often treated by androgen withdrawal In these cases it may be beneficial to the patient to have a gene therapy vehicle harboring high prostate specific gene expression that is not dependent on testosterone

GENE REGULATION: TECHNOLOGY AND APPLICATIONS

Spliceosome Mediated RNA Trans-Spicing Using a LacZ Target Reporter Mouse

Xiaoming Liu,1 M Puttaraju,4 Gary S Mansfield,4 Meihui Luo,1 Ryan Driskell,1 Lloyd G Mitchell,4 John F Engelhardt.1,2,3

1 Department of Anatomy and Cell Biology, The University of Iowa, Iowa City, IA, United States; 2 Internal Medicine, The University of Iowa, Iowa City, IA, United States; 3 Center for Gene Therapy, The University of Iowa, Iowa City, IA, United States;

4 Intronn Inc, Rockville, MD, United States.

The successful treatment of Cystic Fibrosis (CF) requires both functional correction and proper regulation of the Cystic Fibrosis Transmembrane Regulator (CFTR) protein In order to functionally test the in vivo potential of Spliceosome Mediated RNA Trans-splicing (SMaRT) to repair the human CFTR gene, a transgenic animal model expressing the target region of the human gene in the context of a reporter was created Previous studies in human bronchial xenografts that model stem cell reconstitution of airway epithelia have demonstrated that expression of full length CFTR in dividing

or differentiating cells causes a dramatic decrease in the clonal expansion of CFTR expressing cells This suggests that ectopic or overexpression of full length CFTR in human airway stem cells may confer a selective disadvantage in terms of proliferation and/or differentiation We have previously reported that SMaRT can correct the deltaF508 CFTR mutant protein in polarized airway models SMaRT reduced the level of CFTR-mediated developmental toxicity

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

GENE REGULATION: TECHNOLOGY AND APPLICATIONS leading to improved reconstitution of airway stem cells and partial

restoration of CFTR-mediated Cl- permeability At present, the

trans-splicing efficiency in vivo using SMaRT technology is not

known To address transgene delivery and functional correction more

closely, we developed a transgenic mouse that expresses a 3’ mutant

LacZ mini-gene containing a mini-intron-9 of CFTR in the middle of

the coding sequence, splitting the LacZ cDNA into two exons We

also generated recombinant adenoviral and AAV vectors that encode

pre-trans-splicing molecules (PTMs) designed to base-pair with

human CFTR intron 9 and trans-splice in a non-mutant 3’-LacZ

exon PTMs delivered by both adenoviral and AAV vectors were

capable of rescuing beta-galactosidase activity in cell lines harboring

an integrated form of the LacZ mini-gene target Preliminary studies

indicate that the functional correction of b-galactosidase can be

quantified by histochemistry in the lung and skeletal muscle of

transgenic mice after the administration of the PTM by adenoviral

or AAV vector These results demonstrate that SMaRT technology

can efficiently repair a mutant gene by trans-splicing into human

CFTR intron 9, and that PTMs can be delivered in vivo by viral

vectors to the appropriate tissues SMaRT offers the potential to

treat a wide range of diseases caused by genes which are larger than

the <5 kb packaging capacity of AAV and, perhaps more importantly,

to appropriately regulate them by acquiring the expression pattern

of the endogenous target gene

Supported by SBIR grant R44 DK 56526 (LGM) from the NIH

to Intronn and RO1 DK47967 to JFE

Vectors with Low Basal Activity and High

Inducibility for Specific Gene Regulation in the

Liver

Maider Zabala,1 Lin Wang,1 Cheng Qian,1 Jesus Prieto,1 M

Gabriela Kramer.1

1 Internal Medicine, University of Navarra, Pamplona, Navarra,

Spain.

Pharmacological control of gene expression can be achieved by

using gene switch regulatory systems The Tet-on system is

composed of a transcriptional activator protein (rtTA2S-M2) that

binds to a minimal promoter in the presence of doxycycline and

induces gene expression; however it has shown substantial

background activity In order to improve this system, we have

substituted the minimal region of the inducible promoter for a less

active sequence To restrict gene induction to the liver we have used

a set of novel chimeric liver-specific promoters to drive

M2 We have constructed different vectors carrying both the

rtTA2S-M2 controlled by the liver promoters and the luciferase reporter

gene driven by the modified inducible promoter The chimeric

promoters used in this study were those formed by the enhancer II

of the human hepatitis B virus or the enhancer of mouse albumin

gene fused to the promoter of the human a 1 antitrypsin gene, and

the promoter of the human hemopexin gene Activity of these three

promoters has been tested in vivo in a long-term study We have

construct single vectors carrying both transcription units in different

orientation and we observed that the basal and final protein levels

depend on the strength of the promoter that directs the

transcripcional activator as well as the orientation of the two genes

All the resulting vectors allowed liver-specific gene regulation with

lower basal activity and higher inducibility compared to the original

system We have selected one of these vectors to regulate expression

of a transgene in vivo Transfer of the DNA vector to mice was

achieved by using the hydrodynamics-based procedure

Administration of doxycycline enhanced the expression in a

dose-dependant manner, while undetectable levels were observed in the

non-induced status Gene activation could be re-induced even several months after plasmid administration Moreover, sustained continuos expression could be achieved with constant infusion of doxycycline

Specificity Genome Wide

Siyuan Tan,1 Dmitry Guschin,1 Albert Davalos,2 Ya-Li Lee,1 Kaye Spratt,1 Chris Ullman,1 Lindsey Reynolds,1 Michael Moore,1 Casey C Case,1 Carl O Pabo,1 Judy Campisi,2 Philip D Gregory.1

1 Sangamo BioSciences Inc, Richmond, CA; 2 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA.

Designed zinc-finger protein transcription factors (ZFP-TFs) are capable of controlling the expression of any desired target gene, and thus provide potential therapeutic tools for the treatment of disease Here we show that such ZFP-TFs can effectively knock down the expression of a target gene, while providing single-gene specificity

of action within the genome Stable cell lines expressing a ZFP-TF that binds to an 18-bp recognition sequence within the promoter of

the endogenous CHK2 gene were generated by retroviral-mediated

transduction We combined an inducible expression system for

controlling the level of the CHK2 specific ZFP-TF with a

self-inactivating MoMLV-based retroviral vector to generate a broadly applicable delivery vehicle incorporating regulated ZFP-TF expression Amphotropic viruses were produced with the Phoenix high-titer packaging line, and used to transduce HEK293 and U2OS cells Induction of ZFP-TF expression represses the transcription

of CHK2 mRNA, reducing protein levels by >10-fold in both of

these stably transduced lines This level of repression was sufficient

to generate a functional protein knock out, as demonstrated by the loss of the CHK2-dependent activation of p53 induced by DNA-damage The specificity of ZFP-TF driven repression was determined at the mRNA level using microarray technology We

found that just a single gene, CHK2, was regulated by the ZFP-TF

within the monitored genome (22,000 probes) Moreover, this result

is independent of the cell type tested These data demonstrate the utility of ZFP-TFs for target validation and highlight the potential

of these agents as therapeutic treatments in the clinic

Incorporating Retinoic Acid Responsive Elements Provides a Platform for Assessment of

Transcriptional Responsiveness to Retinoids in Cancer Cell Lines

Diana E Jaalouk,1 Laurence Lejeune,1 Milena Crosato,1 Sylvie Mader,2 Jacques Galipeau.1,3

1 Lady Davis Institute for Medical Research, McGill University, Montreal, QC, Canada; 2 Department of Biochemistry, Universite

de Montreal, Montreal, QC, Canada; 3 Division of Hematology-Oncology, Jewish General Hospital, Montreal, QC, Canada.

All-trans retinoic acid (ATRA) has long been known as a transcriptional activator that inhibits cell proliferation and promotes differentiation of many cell types Recently, the drug’s efficacy in anticancer therapy has been broadened by reports suggesting a synergistic effect of ATRA to several cancer gene therapy approaches Our objective is to establish an efficient gene expression system that will provide a screening basis for retinoid responsiveness

in cancer cell lines We hypothesize that cell lines transduced with retrovectors incorporating retinoic acid responsive elements (RAREs) will exhibit modulated transgene expression upon retinoid stimulation To test this hypothesis, we designed a self-inactivating retrovector by creating a 341 bp deletion in the U3 region of the Moloney Murine leukemia Virus 3’LTR We then incorporated into