Figure 2 Types of retroviral vectors.

LTR; ¡3333 thera peutic gene (forward orientation); 5253 therapeutic gene (reverse orientation); S2S33 dominant selectable marker gene; 0 promoter; ® Internal Ribosome Entry Site; retroviral mRNA (forward orientation); *w-«- retroviral mRNA (reverse orientation).

which contain a prokaryotic origin of replication and a prokaryotic selectable marker (AmpR).

Single and multigene retroviral vectors

Single-gene vectors contain one transgene, often transcriptionally regulated by the 5' I.TR, in a retroviral construct. Multigene vectors can also be made since retroviral particles can accommodate up to 7 kb of insert DNA without affecting production of recombinant viral particles. Expression of the upstream gene is usually regulated from the 5' LTR while the downstream genes are expressed from either additional promoters or internal ribosome entry sites (1RES). The advantage of internal promoters is that one can vary expression of the downstream gene by choosing between strong viral promoters for high expression (Rous sarcoma virus (RSV) or cytomegalovirus (CMV)), tissue-restricted promoters for tissue-specific expression (albumin), or inducible promoters for inducible expression

(tetracycline promoters). IRES sequences are derived from picornoviruses (either encephalomyocarditis virus or polio virus), are about 500 bp in length, and allow for ribosomes to initiate translation at internal start codons on polycistronic mRNA. The advantage of IRES sequences is that they make expression of the downstream gene dependent on expression of the upstream gene. A selectable marker gene is often used in this downstream position so that clones isolated after selection for the marker gene are guaranteed to express the upstream therapeutic gene. As many as three genes have been expressed from a single retroviral vector utilizing combinations of IRES and internal promoters. These tricistronic constructs seem to give consistently lower expression than the bicistronic constructs, but whether this is a translation or RNA stability issue remains unclear.

Splicing retroviral vectors

Expressing genes from the 5' LTR promoter can be suboptimal since the transgene is cloned about 600 bp downstream of the transcriptional initiation site. Based on the scanning model for translational initiation, one would predict that this would lead to inefficient translation of the transgene. To minimize this problem, splicing vectors have been engineered in which a partially used splice acceptor sequence is cloned just upstream of the transgene. In the spliced messages, the translational start site of the transgene is brought closer to the 5' end of the message, which should result in higher translation efficiency and expression of the transgene. These splicing vectors give consistently higher expression of transgenes compared to vectors without the splice acceptor.

Reverse orientation vectors

Reverse orientation vectors are constructed by cloning a transcriptional unit (including promoter, cDNA, optimal introns, and poly-A sequences) in the opposite orientation to the retroviral LTR transcript. The primary reason for doing this is that introns can be incorporated in the reverse orientation but not in the sense orientation and intron-containing constructs tend to give higher expression than constructs lacking introns. Introns in the sense orientation would be spliced out in the nucleus of the producer cell before the vector was packaged. Problems with reverse orientation constructs include promoter interference between the LTR and the transgene promoter and antisense effects leading to lower expression and titers. A second problem that has been observed is frequent deletion of all or parts of transgenes from aberrant splicing due to splice sites in the antisense transcriptional unit.

Self-inactivating (SIN) vectors

Two types of vectors were generated predominantly to address early safety concerns: self-inactivating (SIN) vectors and suicide vectors. SIN vectors have a deletion in the 3' LTR enhancer which renders both LTRs transcriptionally inactive in the target cell. These vectors were initially designed for safety reasons to prevent the 3' LTR from promoting or enhancing spurious transcription of a gene adjacent to the site of integration. The specific concern was inappropriate activation of a cellular oncogene. Early concerns about insertional mutagenesis, however, have proven unfounded due to the enormous size of the target genome, so these SIN vectors have not been used extensively. SIN vectors are currently used in reverse orientation constructs to prevent the problem of promoter interference and in cases where an internal promoter is preferred to the 5' LTR.

Double copy retroviral vectors

Double copy vectors are designed to provide two copies of a therapeutic gene in one retroviral vector by inserting the transgene into the U3 region of the 3' LTR. The process of reverse transcription copies this sequence segment such that, when the provirus is formed, the transgene appears in both the 5' and 3' LTRs. Having two copies of the gene should result in higher expression than a single copy, but these vectors have not proven better in most cases.

Retroviral vectors with and without selectable markers

Most of the gene therapy studies and clinical trials have used retroviruses containing dominant selectable marker genes such as drug-resistant markers. The most common selectable marker gene has been the bacterial neomycin phosphotransferase jneo) gene. Selectable marker genes have been used for selecting and characterizing retroviral producer cell clones and for enriching target cell populations for transduced cells. However, investigators have recently suggested that bacterial drug-resistant markers should be eliminated or replaced with natural human proteins less likely to elicit an immune response that would eliminate targeted cells in vivo. The ideal dominant selectable marker would be a human cell surface protein that is not normally expressed on the target cell. Cell surface markers would allow more rapid selection of targeted cells by immunomagnetic selection or FACS compared to drug resistance. Human CD24 and NGFR (p75, nerve growth factor receptor) are examples of human cell surface proteins currently used as dominant selectable markers.

Eliminating selectable markers altogether may have advantages because including two genes in one vector may lead to lower titers or reduced therapeutic gene expression by promoter suppression. For these reasons, many investigators are turning to 'simplified' retroviral vectors which lack dominant selectable markers. Work with simplified vectors had been limited by the laborious methods required to isolate, characterize and titer useful producer clones but recent efforts to simplify this process may encourage greater use of these vectors.

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