875 cytochrome p450 based suicide gene therapy using high titer, expression targeted, retroviral vectors

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

Copyright ®The American Society of Gene Therapy

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RNA VIRUS VECTORS III

particles/ml as determined by EGFP expression in transduced A549

lung carcinoma cell line We then used FLYRD18-EGFP retroviral

particles to transduce human peripheral blood T-lymphocytes that

were isolated from healthy donors by standard Ficoll-gradient

method, then activated with 1μg/ml phytohemagglutinin for ~72

hours in RPMI -10% FBS supplemented with 200U/ml human

IL-2 At the time of transduction, the T-cells were suspended at ~1x106

cells/ml in retroviral supernatant supplemented with 200U/ml IL-2,

and 6μg/ml polybrene, then centrifuged at 600g for ~3hours, at

37°C This procedure was repeated once after 10-12 hours We

ascertained percentage of gene-modified T-cells by flow cytometry

measurement of green fluorescence We had an average of 62 ± 5.5%

GFP+ cells (n=3) We also analyzed gene transfer efficiency in the

CD4 and CD8 T-cell subsets The average of two experiments

revealed that 52.0% of CD4 and 61.1% of CD8 T-cells expressed

GFP, respectively In conclusion, these results demonstrate that

transduction with RD114-pseudotyped retroparticles generated from

FLYRD18 producer cells combined with an optimized spinoculation

protocol can result in stable and high-efficiency gene transfer into

both CD4+ and CD8+ human peripheral blood T-lymphocytes

that is superior to that reported with GALV, amphotropic, or

VSVG-pseudotypes

Third-Generation Lentiviral Vectors

Frank Park

1 Medicine and Pharmacology, Louisiana State University Health

Sciences Center, New Orleans, LA, United States.

There remains a paucity of efficient renal-based gene transfer

methodologies in vivo For this reason, this study has designed and

cloned new third-generation lentiviral vectors to assess the utility of

this system for renal gene therapy Lentivector constructs were

designed and tested for its transduction efficiency in the presence

and absence of important viral accessory genes, such as the rev

responsive element (RRE) Basic lentivectors containing the bacterial

lacZ gene driven by the phosphoglycerokinase (PGK) promoter

were cloned devoid of the RRE in the integrating plasmid

Conditioned medium was collected following quadruple-plasmid

transfection of the HIV split genome into 293T cells, and the viral

titer was assessed by X-gal staining in HeLa cells The basic

lentivector resulted in extremely low titers [less than 10,000

transducing unit (T.U.)/mL; n=5 vector preparations) Incorporation

of a minimal RRE (253 bp) in the 3’ end of the transfer vector

resulted in >100-fold increase in the viral titer to 1.1 +/- 0.4 x 106

T.U./mL (n=5) Interestingly, this increase in viral titer was

position-dependent since the inclusion of the RRE 5’ to the internal PGK

promoter resulted in only ~3-fold higher titer than the vector devoid

of the RRE (n=5) Cloning of the woodchuck post-regulatory element

(WPRE) or one copy of the constitutive transport element (CTE)

from the simian retrovirus type 1 (SRV-1) into the integrating vector

resulted in little enhancement of viral titer compared to the basic

lentivector However, multiple copies of the CTE in the transfer

vector resulted in significant increases of the viral titer to 2.3 +/- 0.4

x 106 T.U./mL (n=5) To further optimize the vector system, manipulations were performed in the packaging construct to replace the RRE with multiple copies of the CTE Efficient expression of p24 Gag protein was detected by ELISA using the modified packaging plasmid and more importantly, viral titer was >3 x 105

T.U./mL This new third-generation vector system is now devoid of all six of the HIV accessory genes (i.e., vif, vpr, vpu, nef, tat and rev), which should theoretically further minimize the potential for replication competency Interestingly, the presence of rev enhanced the viral titer in the lentivectors containing only the multiple copies

of the CTE by ~4-fold (1.3 +/- 0.2 x 106 T.U./mL; n=5)

In vitro screening on the ability of the newly developed

third-generation lentivectors to transduce kidney-derived cell lines from different species was assessed The highest transduction was observed using renal cell lines originating from mice, such as RAG and TCMK-1 whereas 10-fold lower transduction was observed

using both COS-7 (green monkey) and MDCK (canine) In vivo

experiments injecting concentrated lentivectors into the parenchyma

of Sprague Dawley rat kidneys resulted in relatively efficient transduction of epithelial cells using the modified third-generation lentivectors These experiments demonstrate the development of a highly modified lentivector system based on HIV, which can be

utilized as a viable vehicle to deliver transgenes into kidney cells in

vitro and in vivo.

Therapy Using High Titer, Expression Targeted, Retroviral Vectors

Harry Holzmueller,1 Matthias Renner,2 Anika Stracke,1 Michaela Lugauer,1 Brian Salmons,2 Walter H Guenzburg,1 Juraj Hlavaty.1

1 University of Veterinary Medicine, Institute of Virology, Vienna, Austria; 2 Austrianova, Vienna, Austria.

A hallmark for a successful gene-directed enzyme prodrug therapy (GDEPT) is efficient and tumor-specific expression of the prodrug-activating enzyme To meet these requirements a powerful gene delivery system, which enables strong and tumor-restricted gene expression, is needed Therefore, we developed the promoter conversion (ProCon)-vector system, in which the retroviral 3‘LTR U3-region is replaced by a tissue-specific promoter Thus, after infection of target cells the heterologous promoter replaces the viral promoter in the 5‘LTR as a result of reverse transcription and regulates expression of the transgene in these cells Unfortunately, tissue specifity of retroviral vectors often is associated with reduction

in viral titer and/or shut down of transgene expression To overcome these problems, several modifications were introduced into MLV-based retroviral ProCon-vectors, resulting in both 2-3 log increase in viral titer and substantially enhanced expression rates The efficacy

of these improved ProCon-vectors to target therapeutic levels of the prodrug-activating enzyme cytochrome P450 to tumor cells

was then tested in vitro To this end the modified P450-containing

ProCon-vector pPCCWmCMV haboring the murine CMV promoter in place of the 3‘LTR U3-region was constructed Although this promoter is not specific for pancreatic tumor cells, it is strongly active and was thus chosen for proof of principle studies Several pancreatic tumor cell lines were stably infected with this ProCon-vector PCCWmCMV or a conventional retroviral ProCon-vector LXSN carrying the cytochrome P450-gene (LCSN), respectively Western Blot assays demonstrated significantly enhanced P450-expression levels in PCCWmCMV-infected cells when compared to LCSN-infected cells Preliminary data revealed a higher enzymatic activity

of expressed cytochrome P450 and thus a stronger substrate turn-over in tumor cells infected with PCCWmCMV than in cells infected with LCSN as measured by resorufin assays The bioactivity of P450 was evaluated by cell-killing assays showing that

P450-Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

RNA VIRUS VECTORS III

mediated sensitivity of PCCWmCMV-infected tumor cells to

ifosfamide is several times higher than of LCSN-infected cells In

conclusion, efficient killing of pancreatic tumor cells with an

improved ProCon-vector in vitro was demonstrated The in vivo

therapeutic efficacy of these ProCon-vectors is currently under

investigation

to the Liver: Transcriptional Targeting of

Heterologous Genes Depends upon Choice of

Both Promoter and Enhancer

Kathryn L Nash,1 Bushra Jamil,1 Graeme J Alexander,1 Andrew

M Lever.1

1 Department of Medicine, University of Cambridge, Cambridge,

Cambridgeshire, United Kingdom.

Efficient gene therapy is likely to require long-term, organ-specific

therapeutic gene expression Lentiviral vectors based on HIV-1 are

promising gene delivery vehicles due to their ability to integrate

transgenes into non-dividing cells Transcriptional targeting to the

liver can be achieved by using liver specific promoters This study

compared gene expression from different promoters in tissue culture

cells from hepatic and non-hepatic origin The abilities of different

promoters to direct gene expression following transfection (when

the transgene occupies an episomal location) and transduction (when

the transgene is integrated into the cellular DNA) were also compared

Replication deficient HIV-1 based vectors were produced by

transient co-transfection of 293T cells with vector and helper

constructs Cultured hepatocytes were transduced with vectors

expressing green fluorescent protein (GFP) under the control of the

human Cytomegalovirus (CMV) promoter Transduction efficiencies

exceeding 50% were obtained Gene expression persisted for over 3

months in transduced cells

The CMV promoter in the vector construct was substituted by

the alpha-1 antitrypsin gene promoter or a promoter derived from

the hepatitis B virus genome (HBV) All of these promoters gave

high level GFP expression when transfected into cultured hepatic

and non-hepatic cells Upon hepatocyte transduction, however, only

the CMV, alpha-1 antitrypsin and HBV core promoters were able

to produce GFP expression Addition of HBV enhancer elements

improved the transducing ability of the HBV surface promoter and

increased its specificity for hepatocytes

This study demonstrates that lentiviral vectors can be used to

obtain long-term transgene expression in liver cells The results

indicate that both the promoter and enhancer have to be specific to

achieve high level, liver specific gene expression Furthermore, the

site of the transgene appears to influence the tissue specificity of

the transcriptional control elements suggesting that tissue-specific

promoters must be fully analysed in the context of the vector that is

to be used for their delivery

Human Bone Marrow Mesenchymal Cells

An Van Damme,1 Marinee Chuah,1 Francesco Dell’Accio,2

Cosimo De Bari,2 Frank Luyten,2 Luigi Naldini,3 David Lillicrap,4

Antonia Follenzi,3 Désiré Collen,1 Thierry VandenDriessche.1

1 Center for Transgene Technology and Gene Therapy, University

of Leuven, Leuven, Belgium; 2 Laboratory for Skeletal

Development and Joint Disorders, University of Leuven, Leuven,

Belgium; 3 Laboratory for Gene Transfer and Therapy, University

of Torino Medical School, Italy; 4 Department of Pathology,

Queen’s University, Kingston, Canada.

Human bone marrow (BM) mesenchymal cells are attractive targets

for ex vivo gene therapy This BM-derived cell population contains

adult stem cells capable of self-renewal and differentiation into distinct

mesenchymal lineages In this study we examined the potential of using HIV-1 derived lentiviral vectors for transducing BM mesenchymal cells For comparison of transduction efficiency, a retroviral SIN-vector was constructed, containing the same CMV-GFP-WPRE expression cassette as in the lentiviral vector Human

BM mesenchymal cells were transduced with both vectors at varying MOI’s Transduction efficiency, as measured by FACS analysis, was consistently higher when lentiviral vectors were employed at all MOI’s tested Transduction efficiency could be increased further

by including a centrifugation step in the tranduction protocol Maximal transduction efficiency with the lentiviral vector was 90% (MOI 140), as compared to only 17% with the retroviral vector The increased percentage of GFP-positive BM mesenchymal cells following lentiviral gene transfer versus onco-retroviral transduction was consistent with a concomitant increased gene transfer efficiency and increased GFP mRNA expression We also examined whether transduction with lentiviral vectors influences differentiation of human BM mesenhymal cells into adipocytes and osteocytes in vitro and whether transgene expression is maintained Human BM mesenchymal cells that were nearly 100% GFP positive following transduction with the HIV-CMV-GFP vector, were still capable of differentiating into adipocytes and osteocytes in vitro Upon differentiation, GFP expression in adipocytes as well as in osteocytes persisted When human BM mesenchymal cells were transduced with a lentiviral vector containing a therapeutically relevant transgene (B-domain deleted canine FVIII), adipogenic and osteogenic differentiation was equally efficient as in non-transduced cells FVIII expression in vitro was not statistically different between osteocytes and undifferentiated cells, and was slightly higher in adipocytes than in undifferentiated cells (p=0.04) Hence, in vitro differentiation

of lentivirally transduced human BM mesenchymal cells does not lead to transcriptional down-regulation These results indicate that lentiviral vectors are well suited for transduction of BM mesenchymal cells

MGMTP140K Protects Human CD34+ Cells Against

BCNU or Temozolomide

Dhanalakshmi Chinnasamy,1 James Neuenfeldt,1 Leslie Fairbairn,2

Geoffrey Margison,3 Nachimuthu Chinnasamy.1

1 Vince Lombardi Gene Therapy Laboratory, Immunotherapy Program, St Luke’s Medical Center, Milwaukee, WI; 2 Gene Therapy Group, Paterson Institute for Cancer Research, Manchester, United Kingdom; 3 Carcinogenesis Group, Paterson Institute for Cancer Research, Manchester, United Kingdom.

O6-alkylating agents such as BCNU and temozolomide are commonly used cancer chemotherapeutic agents, but their usefulness

is limited by dose-limiting myelotoxicity mainly due to the low

level of expression of O6-alkylguanine DNA alkyltransferase

-benzylguanine (O6-beG), which is undergoing clinical trials, potentiates alkylating agent induced cytotoxicity in tumors by inactivating MGMT but also increases toxicity in hematopoietic cells It has been shown that retroviral-mediated expression of mutant forms of MGMT can protect hematopoietic progenitor cells against

the combined toxicity of O6-beG and BCNU or temozolomide However, murine leukemia virus-based retroviral vectors do not transduce nondividing hematopoietic stem cells Accumulating evidence suggests that lentiviral vectors are capable of transducing nondividing cells including quiescent hematopoietic stem cells We constructed self-inactivating HIV-1 based lentiviral vectors expressing wild type and mutant MGMTP140K cDNA driven by the human PGK promoter A three-plasmid expression system was used to generate vesicular stomatitis virus (VSV-G) pseudotyped