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drug delivery reviews

ELSEVIER Advanced Drug Delivery Reviews 17 (1995) 235-238

Novel vectors for gene therapy

David S. Strayer* Department of Pathology. Anatomy and Cell Biology, Jefferson Medical College, 1020 Locust Street, Philadelphia. PA 19107.

USA

Accepted June 1995

Abstract

A novel viral vector for potential use in gene transduction is described. It takes advantage of a powerful endogenous promoter, a wide range of potential animal hosts and cellular targets, the power to mediate stable gene expression, and the ability to be concentrated to high titer. We used a replication-deficient variant of SV40 to mediate gene transfer in vitro and in vivo. Mutant virus containing luciferase reporter gene (SVluc) transferred the ability to produce enzymatically active luciferase. This luciferase was detected by immunohistochemistry and enzymatic assay. Mice inoculated intravenously or intratracheally with SVluc expressed luciferase in multiple organs including liver, lungs, spleen, and skin. Luciferase expression has been stable to 3 weeks post inoculation, the last time point examined. Mice showed no evidence of immune elimination of luciferase-expressing cells. Replication-deficient SV40 may be a useful addition to the current list of viral agents applicable to gene therapy.

Keywords: Gene therapy; Transduction; Virus infection: Gene expression

Contents

1. Experimental approach, methods and results ................................ 1.1. sv40 ................................................................................... 1.2. Constructing mutant SV40 ............................................................... 1.3. Testing for functional activity in vitro: production of replication-deficient SV40 containing IacZ

1.4. Testing for functional activity in vitro: construction of mutant SV40 containing luciferase ...... 1.5. Testing for functional activity in vivo: SVluc inoculated into mice ............................

2. Discussion and future directions ............... ..............................................

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1. Experimental approach, methods and results

Currently available approaches to transducing foreign genes include both viral and nonviral gene transfer agents. Among the viral agents used for these purposes and retroviruses, adeno- virus, and adeno-associated virus. Nonviral

* Tel. (215)923-7689; Fax (215)923-2218.

agents principally include liposomes of various kinds. Each of these transduction agents has strengths and limitations. Immune responsive- ness may limit the usefulness of one agent, inability to be concentrated to adequate titer another, etc. While one vector may be best suited to a particular situation, none of these vectors is clearly superior to the others for general use. Thus, there is ample room to accommodate

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novel, previously unreported approaches to gene therapy.

1.1. sv40

The potential of SV40 as a gene transfer agent lies in several observations. The virus in its wild- type (wt) form, was released into the human population inadvertently as a contaminant in early Salk polio vaccine, without ill effect. Fur- ther, SV40 has a wide infectivity both for differ- ent host organisms and cell types. The virus can be concentrated to high titer, and is associated with high levels of gene expression and integra- tion into target cell genome. The viral genome is small enough that it can be cloned into a plas- mid, which simplifies its manipulation. SV40 is easy to grow, and many cell lines are available that express part of the SV40 genome; so that replication defective viruses can be propagated easily.

The oncogenic potential of SV40 mainly re- flects its ability to immortalize cells in vitro, and is reported in vivo only in very limited settings, SV40-induced transformation is generally consid- ered to be a function of its large T antigen (Tag), which binds both ~53 and pRb, and which by itself may confer transformed phenotype on transfected cultured cells. We therefore reasoned that replication-deficient SV40 virus from which Tag had been excised could represent a reason- able and relatively safe approach to gene trans- fer.

1.2. Constructing mutant SV40

We began with a plasmid construct in which the SV40 genome was cloned into pBR322, and then redesigned the viral genome in this plasmid to delete Tag. This yielded a viral genome incapable of replicating by itself. To permit both virus generation and propagation, a cell line constitutively expressing Tag was used. In place of Tag, we inserted an engineered multiple cloning site (mcs), located immediately down- stream from the SV40 early promoter. To facili- tate cloning, this plasmid construct permitted easy propagation in E. coli.

1.3. Testing for functional activity in vitro:

production of replication-deficient SV40

containing 1acZ

Our first step was to determine whether this construct be used to generate replication de- ficient virus particles. We cloned the gene for the E. coli operon protein 1acZ into the mcs and transfected the resulting construct into two dif- ferent cell lines. We determined by RT-PCR that 1acZ was expressed in these transfected cells.

Next, we ascertained that the construct could be used to generate virus particles and that these virus particles were replication deficient. The SV40 genome containing 1acZ was excised from pBR322, recircularized, and transfected into Tag- expressing COS-7 cells. Shortly after transfec- tion, cytologic changes were observed, consistent with those seen during replication of wild-type SV40. Resulting virus preparations could be titered using Tag-supplying COS-7 cells but not using TC7 cells, which do not supply Tag. This finding indicates that the SV40 (1acZ) recombi- nant virus was replication deficient and that when cells provided the lacking viral protein (Tag) replication could proceed.

1.4. Testing for functional activity in vitro:

construction of mutant SV40 containing

luciferase

Our next step was to determine whether this construct could transfer to target cells the ability to produce a protein that could be recognized by immunohistochemical means, and whose activity could be assayed functionally. For this purpose, we used firefly luciferase (luc). The gene for luciferase was cloned into the mcs (together with a second copy of the SV40 early promoter). The SV40 genome was excised from the resultant plasmid and transfected into COS-7 cells to produce replication deficient SV40 viruses con- taining luciferase (SVluc).

Resultant SVluc were used to infect TC7 and COS-7 cells in culture. These cells were assayed for luciferase protein by immunocytochemistry with affinity-purified anti-luciferase antibody (Promega), and for luciferase enzymatic activity using a standard light emission assay, supplying luciferin and ATP, which are required for the

D.S. Strayer I Advanced Drug Delivery Reviews 17 (1995) 235-238 231

production of light by active luciferase enzyme vivo. Using luciferase as a reporter gene, we (Promega). We found that the infected cells constructed replication-deficient SV40 viruses stained strongly positively for luciferase protein that can cause target cells to produce the firefly and that they displayed strong luciferase activity enzyme luciferase to such levels that it can be as determined in a luminometer. Control cells, detected by enzymatic assay in whole organ treated with vehicle alone, produced no detect- homogenates at least as long as 21 days after able light. inoculation in vivo.

1.5. Testing for functional activity in vivo: SVluc inoculated into mice

To determine whether SVluc transferred luciferase activity to animals receiving this re- plication-deficient SV40 mutant, we inoculated lo9 SVluc into BALB/c mice, either intraven- ously or intratracheally. Mice were assayed from 1 to 21 days after infection for cellular expression of luc by immunohistochemistry using frozen sections of selected organs. Luciferase activity was also determined using whole organ homoge- nates from SVluc infected mice.

These data, up to 21 days, do not represent a long enough or large enough study to permit unambiguous assessment of the longevity of SV40 mediated gene expression. However, the flat but clearly detectable enzymatic activity curve up to that point suggests that cautious optimism may be appropriate regarding the po- tential utility or applicability of this vector.

By immunohistochemical analysis, luc protein was detected at all time points following inocula- tion by either route. Frozen sections of liver, spleen, heart, lungs, kidneys and skin at the inoculation site were studied using anti-luciferase antibody. In the liver, both hepatocytes and Kupffer cells produced immunologically detect- able luciferase protein. In the lungs, bronchial lining cells stained positively for luciferase. Skin at the site of inoculation was also positive, as were lymphocytes in the spleen, proximal tubular cells in the kidney, and keratinocytes in the skin. There was no evidence of immunologically me- diated destruction of luciferase-positive cells. Nor was there evidence of an ongoing cellular immune response in the organs studied.

One of the problems of adenoviral transfer vector is the relatively short duration of gene expression, owing in part to immune responsive- ness against the virus-infected cells. Although we have not yet conducted experiments involving mice inoculated more than 21 days prior to assay, we did not observe any evidence of immunologi- cally mediated cytolytic activity in SVluc-infected mice, nor did enzymatic activity appear to de- cline at that time point. Rather, both organ histology and the time course of luciferase activi- ty assayed in whole organ homogenates sug- gested that a steady level of activity had been attained and that the luciferase-producing cells had to that point been accepted by the host.

One of the difficulties encountered when retro- viral transfer vectors are used is the fact that retroviruses do not survive concentration well, and that very high infectious viral concentrations cannot be maintained. This is not a problem with SV40. For the most part, band-purified SV40 can be prepared at titers of >109-10 virus/ml.

Enzymatic assay of whole organ homogenates for luciferase activity showed no detectable ac- tivity until 1 week following inoculation. There- after, activity was detected, principally in the liver, kidney and skin at the inoculation site. Expression was stable throughout the 21 day study.

2. Discussion and future directions

Thus, SV40 can effectively transfer foreign genes to mammalian cells, both in vitro and in

Our data are preliminary. They are in need of considerable additional study to determine the longevity of expression. SV40 transfer vectors must be further adapted to use genes of potential therapeutic utility, e.g., CFTR. We have per- formed very preliminary studies that document our ability to transfer human CD4 s a cell membrane protein to TC7 monkey fibroblasts. But much more information is needed to assess the degree to which this vector can be used to cause usable gene transfer in a therapeutic set- ting.

There are also potential limitations to the

238 D.S. Strayer I Advanced Drug Drlivrry Review I7 (I 9W) 235-2.3X

system that should be evident at the outset. Unlike adenovirus, SV40 is a very small virus. Excising Tag provides 2.2 kb in the genome. The luciferase construct we used was about that size, and functioned well. The 1acZ construct was 0.3 kb, and also functioned well. However, the size limitations of this construct, particularly the upper limit of size, are not known. Additional studies must be done to delineate the size range of inserts acceptable to the virus in this system.

Further, SV40 has little inherent tissue trop- ism: it may infect a wide range of target tissues. The issue of specificity must, then, be addressed. Promoters other than the endogenous SV40 promoter will have to be studied to see to what extent tissue specificity in expression can be obtained by promoter specificity.

Studies in other animal species, more complete examination of organs for gene expression, and more extensive subcellular protein localization

studies must be done. It is not known, for example, whether SV40 vectors can produce proteins that localize in the nucleus. Neither is it certain that this vector will necessarily mediate the gene expression in humans.

The safety of this vector must be established. Presumably, it is safe: large scale inoculation of people with wt SV40 occurred inadvertently with little apparent adverse effects. The principal theoretical concern, the presence of Tag, is largely eliminated by using Tag-deficient mutants remains. The potential for incorporation of Tag from COS-7 cells must be determined.

Despite these caveats, and the clear need for much more study of this system, our data to this point strongly suggest that SV40 is an effective gene transfer agent. It is hoped that it will be a therapeutically useful addition to the growing list of gene transfer vectors.