533 lentiviral vectors mediated long term and high efficiency expression of foreign gene in HEK 293t cells

533 Lentiviral Vectors Mediated Long Term and High Efficiency Expression of Foreign Gene in HEK 293T Cells Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene[.]

Molecular Therapy Volume 21, Supplement 1, May 2013

In conclusion, our current results provide a novel chemoselection

strategy using shRNAs to increase engraftment of genetically

modifi ed HSPC so that it may be possible to use this technology for

the stable control of HIV

531 Designing Trans-Regulated Lentiviral

Vectors for Gene Transfer in Functional Genomics

and Gene Therapy

Agnes Holczbauer,1 Adriana Zingone,1 Suresh Arya.2

1 Center for Cancer Research, National Cancer Institute, Bethesda,

MD; 2 Center for Cancer Research and Division of Cancer

Treatment and Diagnosis, National Cancer Institute, Bethesda,

MD.

One defi ciency in effi cient functional genomics analysis and

mounting successful gene therapy trials is the paucity of a vector

system that can be regulated from within or without Knowledge of

the regulation of lentivirus HIV gene expression presents a possible

strategy The Tat gene of lentiviruses encodes transactivator protein

that regulates viral gene expression by acting through the viral

promoter We are using this observation to design a trans-regulated

lentiviral vector system for gene transfer For a number of reasons,

we are using vectors derived from both HIV-1 and HIV-2 that could

be used to create hybrid or chimeric vectors with additional safety

insurance The distinctive feature of this system as compared with the

conventional vectors is that the transfer vector does not include an

internal promoter to drive transgene or shRNA expression Instead,

the expression is driven by the viral LTR promoter itself The viral

promoter is essentially silent in most cells and needs Tat for activation

This provides the means to regulate vector expression in trans The

model vector must fulfi ll the following requirements: (i) when

introduced into target cells, it must display low basal activity, (ii) if

Tat is provided genetically, the target cell must be able to synthesize

suffi cient Tat, and if Tat is provided as a protein, it must reach the

nucleus of the target cell with its integrity intact, and (iii) the vector

must able to respond to Tat to activate transgene expression Because

of the possible use of this system in cancer gene therapy, we tested

the system in a number of cell lines from the NCI 60 cell panel that

included cell derived from major solid tumors and lymphoblastoid

cells We used GFP as a reporter gene and provided Tat genetically

in trans For the twenty cell lines tested, by and large the model held

There was low but variable level of basal GFP expression depending

on the cell line, and it was elevated by Tat in all cell lines but again

to variable extent (5 - 20 fold) This elevation was not observed

with the unrelated puromycin vector As an alternative to providing

Tat as a gene or protein, one conceivably could fi nd a small

drug-like molecule by high throughput screening of chemical libraries

We have developed a cell-based assay towards this goal, where

transgene expression is dependent on Tat, which could be used for

library screening The opinions expressed in this abstract are those

of the authors and do not necessarily represent views of the National

Cancer Institute

Nanovesicles (Gesicles) for the Delivery of Active

Protein To Target Cells

Thomas P Quinn,1 Lily Lee,1 Mei Fong,1 Michael Haugwitz,1

Hiroaki Sagawa,2 Andrew Farmer.1

1 Clontech Laboratories, Inc., Mountain View, CA; 2 Takara Bio

Inc., Otsu, Shiga, Japan.

Historically, phenotypic control of live cells has been achieved

through transient or stable delivery of nucleic acids into primary or

transformed cells However, this approach has a number of drawbacks,

including low effi ciency, toxicity, genome disruption at the integration

site, and a potentially signifi cant delay in phenotypic response due

to the requirement for transcription and translation post-delivery Another approach is to use an inducible system which allows for tight regulation of expression—but inducible systems still depend upon effi cient delivery of DNA to function properly In contrast, direct intracellular delivery of recombinant proteins into live cells allows tight control over both the timing and dose of the desired polypeptide, but is limited by the need to express the protein prior

to delivery (typically in E coli) This can lead to issues with protein

yield, proper folding, and post-translational modifi cations, and likely

to a reduction in activity Alternative methods of protein delivery could provide new and effective tools Here we report on a number of applications that utilize VSV-G-induced microvesicles (Gesicles)1 to deliver functional proteins directly to target cells Co-overexpression

of the spike glycoprotein of VSV-G with the POI, within a mammalian packaging cell, leads to the production of VSV-G coated microvesicles containing active amounts of the POI Expanding upon previous work1, we have developed a number of Gesicles carrying the receptors Pit2, mCAT1, and CAR to alter the susceptibility of primary cells and cell lines to various viruses, including retroviral and lentiviral vectors pseudotyped with either amphotropic and ecotropic envelopes as well

as type 5 adenovirus vectors In all cases, transduction of receptor Gesicle-treated cells resulted in an increase in transduction effi ciency, including rendering receptor-negative cells positive for transduction

as measured by fl uorescence microscopy and FACS We were also able to deliver other functional membrane proteins such as GPCRs and a cell surface marker, with both demonstrating activity in as little

as 3 hours post delivery To our knowledge, no other current delivery method is capable of delivering membrane proteins in this way Other experiments confi rmed that we could deliver fl uorescent proteins and other molecules to the cytoplasm All activities were found to be due

to direct transfer of the POI contained within the Gesicles and not a byproduct of indirect nucleic acid transfer This work suggests that Gesicles can be considered as another effi cient means to direct the phenotype of a cell of interest through the rapid and direct delivery

of active protein to a chosen target cell Reference: 1 Mangeot et al (2011) Mol Ther 19:1656-1666

and High Effi ciency Expression of Foreign Gene in HEK 293T Cells

Renhe Yan,1,2 Yingying Mao,1 Zhibing Liang,1 Yi Feng,2 Tianbai

Li,3 Maomin Lu,4 Weiwang Gu,2 Jingang Zhang,4 Hongwei Li.1

1 School of Biotechnology, Southern Medical University, GuangZhou, GuangDong, China; 2 Institute of Comparative Medicine and Center of Laboratory Animals, Southern Medical University, GuangZhou, GuangDong, China; 3 College of Medicine, University of Florida, Gainesville, FL; 4 Institute of Transfusion Medicine, The Academy of Military Medical Sciences, Beijing, China.

Objectives: Lentiviral vectors have been developed to successfully

and rapidly produce decigram quantities of active recombinant proteins in human cell lines To optimize the protein production platform, the roles of different promoters, insulator, and Ubiquitous Chromatin Opening Element (UCOE) were evaluated in the effi ciency and stability of foreign gene expression mediated by lentiviral

vectors Methods : Five lentiviral vectors,

pFIN-EF1-GFP-2A-mCherH-WPRE containing EF1 promoter and HS4 insulator, pTYF-CMV(-globin intron)-EGFP containing CMV promoter and -globin intron, pTYF-CMV-EGFP containing CMV promoter, p’HR.cppt.3’1.2kb-ucoe-SFFV-GFP containing SFFV promoter and UCOE , and pTYF-EF1-EGFP with EF1 promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell) The transduced cells were passaged once every three days at

a ratio of 1:10 Expression of the foreign gene, GFP, was detected

using fl uorescent microscopy and fl ow cytometry Results: GFP

Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy S206

was stably expressed for at least 9 weeks without signifi cant change

in fl uorescent signal intensity in cultures transduced with the fi ve

different viruses At fi ve weeks post transduction, the positive rate

of GFP expression mediated by lentiviral vectors listed in Methods,

are 22.73%, 54.13%, 57.63%, 6.17% and 38.73%, respectively; while

the mean fl uorescent signal intensities were 6392, 21486, 26597,

1436, and 9468, respectively Conclusion:The results suggest that

all fi ve vectors can stably transduce 293T cells resulting in long

term expression with different effi ciencies Furthermore, insulator

and UCOE do not cause any improvement in GFP expression The

vectors containing the promoters CMV or CMV (-globin intron)

which elicited the highest GFP expressions can serve to create vectors

for protein production

534 Tumorigenic Effect of Transplanted

Myoblasts Overexpressing Bcl-2 Based on

Retroviral Vector

Francisco Martinez-F,1,2 Maria Ustoa,1 Araceli Barrera-L,1 Hugo

E Sandoval-Zamora,1 Bruno Guevara,1 Pablo Vizcaino-Dorado,1

Alejandro Zentella-Dehesa,3 Catalina Machuca-R.4

1 Molecular Biotherapeutic Program, Skin & Tissue Bank, National

Institute of Rehabilitation Ministry of Health, Mexico City,

Mexico; 2 Dapartment of Pharmacology, School of Medicine,

National Univeristy of Mexico, Mexico City, Mexico; 3 Department

of Bichemistry, IIB at INCMNSZ, Mexico, DF, Mexico;

4 Laboratory of Molecular Therapy, FES-Zaragoza, National

University of Mexico, Mexico City, Mexico.

Introduction: The Bcl-2 overexpression confers cytoprotection and

enhances cell resistance in several kinds of cell and tissues Previously

we found that the myoblasts transformed with a retroviral vector to

overexpressing bcl-2 protein in model of muscular reparation, results

in cytoarchitecture preservation and an enhancement of myotubes

differentiation However, the retroviral transformation provides too

a potential tumorigenic factor Objective: To evaluate the tumorigenic

effect of the repaired muscle with transformed myoblast cells with a

retroviral vector to overexpressing bcl-2 in a in vivo model Material

and Methods: The following groups (n=4) of nude mice were

transplanted with 2X106 cells Group A with normal cells (control);

Group B, myoblasts transformed with a retrovirus vector based on

MLV system with CMV-GFP cassette (GFP group); and Group C,

myoblasts transformed with retrovirus containing Bcl-2 (under Pol I

promoter) All animals were maintained in the animal care facilities,

under controlled environment and normal diet After 4 months,

animals were sacrifi ced and studied for molecular and histological

analysis Results: Animals of the control group show macroscopical

rates of fi brosis and atrophy One animal shows a tumour around

the mouth The GFP group shows an increased tumor growth in the

site of transplant and one tumour on the shoulder The Bcl-2 group

shows similar size with the contralateral leg The molecular analysis

of RT-PCR shows a minor expression of Bcl-2 levels related to the

GFP expression The histological analysis was similar to histological

patterns obtained during the day 30 The data shows that the effect

of bcl-2 could improve the control of differentiation and possibly its

implication on the growth process of myoblasts Additionally, this

conserved effect could be a consequence of the different promoter,

suggesting that the low levels of bcl-2 in muscle improve its biological

effect and a tumor phenotype negative Acknowledgments: This

research project is granted by the National Council of Science and

Technology of México and the Ministry of Health of Mexico Grant

FOSIS/CONACYT-Salud-2011-1-161624

535 Transduction of Ferret Airways with Avian Infl uenza Virus Hemagglutinin Pseudotyped Equine Infectious Anemia Virus Vector

Ziying Yan,1 Manij Patel,2 Xingshen Sun,1 Diana CM Lei-butters,1

Hongshu Sui,1 John C Olsen,2 John F Engelhardt.1

1 Department of Anatomy and Cell Biology, Center for Gene Therapy, The University of Iowa, Iowa City; 2 Department of Medicine, The University of North Carolina, Chapel Hill.

Cystic fi brosis (CF) is an inherent disease caused by a single gene defect in the cystic fibrosis transmembrane conductance regulator (CFTR), which causes persistent bacterial lung infections The recently developed CF ferret model rapidly acquires bacterial lung infections similar to the human disease and is an ideal model for testing lung-directed gene therapies for CF To this end, we sought to develop a lung gene therapy strategy that would allow for complementation of CFTR in newborn CF ferrets Since lung epithelial cells are rapidly dividing in the neonatal period, we hypothesize that a lentiviral vector approach would best provide long-term persistent transgene expression in this model Like humans, ferrets are very susceptible to infl uenza virus infection and thus we hypothesized that hemagglutinin (HA) from avian infl uenza A virus (subtype H7) might be ideal for mediating lentiviral infection in the airway of newborn To this end, we pseudotyped equine infectious anemia virus (EIAV) with H7-HA derived from the parental avian infl uenza A stain A/FPV/Rostock/8/34 The reporter vector used in this study was HA-EIAV.Sin6.1CBnZW and encoded a nuclear located lacZ gene driven by CMV enhancer and chicken -actin promoter HA-EIAV.Sin6.1CBnZW titers, as determined on 293 cells, were 2.5x108 IU/ml We fi rst tested infectivity of this recombinant virus

on polarized adult ferret primary airway epithelia (FAE) cultured at

an air-liquid interface (ALI) with an inoculum of 1x106 IU at an MOI

1 IU/cell Results from these studies demonstrated 40-50% cells were transgene positive following basolateral infection However, following apical infection transduction was much less effi cient with

5% LacZ positive cells In vivo airway infection was conducted by intratracheal injection of 7.5x106 IU of HA-EAIV.Sin6.1CBnZW into newborn ferret kits at 2-days of age Two-weeks after infection, tracheas and lungs were harvested and stained with X-gal Grossly, signifi cant transgene expression was seen in all lobes and the large and small conducting airways of the lungs, but not in the trachea Histologic analysis demonstrated that HA-H7-EIAV virus effi ciently transduced bronchi, bronchioles, and alveoli (ranging from 10-70%

of cells in these structures) X-gal positive cells in the tracheal airway epithelia were also detected at a much lower frequency These results suggested the HA-H7 pseudotyped EIAV vector effectively transduce intra-lobar airways of the newborn ferret and may be a good gene transfer agent to test gene therapies for CF lung disease in this model

AAV Vectors III

536 Multiple Molecular Alterations in Phosphodegrons 1-3 within AAV2 Capsid Demonstrates Higher Hepatic Gene Transfer Effi ciency

Dwaipayan Sen,1 V Kalaivani,1 Rupali Gadkari,2 G Sudha,2 N Srinivasan,2 Alok Srivastava,1,3 Giridhara R Jayandharan.1,3

1 Hematology, Christian Medical College, Vellore, India;

2 Molecular Biophysics Unit, Indian Institute of Science, Bengaluru, India; 3 Center for Stem Cell Research, Christian Medical College, Vellore, India.

The success of AAV2 mediated hepatic gene transfer in human trials for diseases such as hemophilia has been hampered by a combination

of low transduction effi ciency and a robust immune response directed against these vectors We have previously shown that AAV2 is