Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2002-03-19
2004-10-19
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S455000, C435S456000, C435S457000, C435S325000, C536S023100, C536S023720, C536S024100
Reexamination Certificate
active
06806080
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of medicine and in particular to vectors for delivery of nucleic acids into cells and to vectors useful for gene therapy.
BACKGROUND
One of the foremost obstacles to the practical implementation of human gene therapy is the lack of an optimal method for the direct delivery of therapeutic genes to quiescent tissues in vivo. A number of vector systems based on viral components have been developed; however, of these individual virus vector systems, none is optimal and each system displays significant drawbacks.
Retroviruses as vehicles for the delivery of genes into eukaryotic cells have several advantages (Hwang and Gilboa, 1984; Varmus, 1988): 1) gene transfer is relatively efficient; 2) stable integration into the host cell DNA is a natural part of the retroviral life cycle, and therefore the integrated provirus is passed on to all daughter cells, and continues to direct the nonlytic production of its encoded products; and 3) replication-defective vectors can be created by deletion of essential viral genes, which renders the vectors incapable of secondary infection (Mann et al., 1983; Markowitz et al., 1988; Miller and Buttimore, 1986). In spite of these advantages, retroviral gene transfer in its current form has several drawbacks. Most retroviral vectors in current use are traditionally based on Moloney murine leukemia virus (MLV), which requires cell division during infection so that the nucleocapsid complex can gain access to the host cell genome, and hence cannot infect non-dividing cells (Mulligan, 1993; Varmus, 1988). Many cell types are considered to be largely quiescent in vivo, and furthermore, most retroviral vectors are produced from packaging cells at titers on the order of only 10
6-7
colony-forming units (cfu) per ml, which is barely adequate for transduction in vivo. Therefore, retroviral gene transfer in vivo is inefficient, and the traditional approach which has been adopted for retroviral vectors has been to transduce primary cells in culture by the ex vivo method, followed by re-implantation of the transduced cells. This approach requires surgical acquisition, isolation, and culture of autologous cells, and thus is labor-intensive and invasive, and limits the scope of ex vivo retroviral gene transfer to those cell types that can be readily accessed, maintained and manipulated in culture, and reimplanted, e.g., hematopoietic cells, skin fibroblasts, and hepatocytes.
On the other hand, adenoviral vectors have been shown to efficiently infect many cells types in vivo by direct injection. However, as the adenoviral vector remains episomal and does not integrate into the host cell genome, transgene expression is transient. The utility of adenoviral vectors is further limited by cellular and humoral immune responses against wild type adenovirus gene products, which appear to be expressed at low levels in the transduced cells due to “leaky” expression despite deletion of the E1 regulatory region (Engelhardt et al., 1993; Yang et al., 1995). Once sensitized, a neutralizing antibody response usually precludes repeat administration by the same vector, and adenovirus-infected cells are soon eliminated by cytotoxic T lymphocytes after transduction (Roessler et al., 1995; Yang et al., 1995). Thus, neither type of virus vector can achieve efficient and long term transduction by direct injection in vivo.
Another virus vector which has been considered is the adeno-associated virus (AAV) (Flotte et al., 1993). AAV was initially thought to be advantageous because it appeared to efficiently infect non-dividing cells (Flotte et al., 1994), and would also undergo site-specific integration into the host cell genome, resulting in long term transduction. However, although these do appear to be attributes of wild type AAV, it seems that these characteristics may not be associated with replication-defective AAV vectors, from which the AAV structural genes, especially the rep gene, have been deleted (Halbert et al., 1995). Other disadvantages of the AAV system have been the limited packaging capacity, only about 4 kilobases, of the vector, and the difficulty of making high titer AAV stocks.
Retrotransposons are mobile genetic elements that insert into new genomic locations by a mechanism that involves reverse transcription of an RNA intermediate. Among the most well-characterized human retrotransposons are L1 elements or LINEs (long interspersed nuclear elements); these non-LTR elements are present in approximately 100,000 copies in the human genome, although 97% of these are functionally inactive due to truncations and rearrangements, and of the remaining 3000 or so full length L1 elements (Singer et al., 1993), it has been estimated that only about 1.5-2.5%, i.e., 30 to 60 copies, are active in retrotransposition (Sassaman et al., 1997). A 6 kb L1 consensus sequence has been derived by sequence analysis of multiple elements (Scott et al., 1987), containing a 5′ untranslated region with an internal promoter (Minakami et al., 1992; Swergold, 1990), two non-overlapping reading frames (ORF 1 and ORF 2), a 3′ untranslated region and 3′ polyadenylated tail; ORF 1 encodes a 40 kD nucleic acid binding protein that co-localizes with L1 mRNA in a cytoplasmic complex (Hohjoh and Singer, 1996; Holmes et al., 1992), while ORF 2 encodes a protein with reverse transcriptase (RT) activity (Hattori et al., 1986; Xiong and Eickbush, 1990) and an N-terminal endonuclease (EN) domain (Feng et al., 1996). Recently, it has been demonstrated that a reporter cassette, with a selectable marker gene driven by the SV40 promoter, can be inserted in reverse orientation into the 3′untranslated region of L1 elements, and when transfected into cells as an EBNA/oriP-containing episomal plasmid, this system can be used to detect retrotransposition events (Moran et al., 1996; Sassaman et al., 1997). The human L1/reporter element was also active in mouse fibroblasts, suggesting that cellular factors involved in retrotransposition are conserved (Moran et al., 1996). Furthermore, this system was used to characterize novel human L1 sequences that were screened from a genomic library; one of these, L1.3, retrotransposed at a considerably higher frequency, about 1 retrotransposition event scored per 150 cells containing the episomal plasmid (Sassaman et al., 1997). In fact, the actual frequency is probably even higher, as the assay system scored only retrotransposition events occurring in cells that had been pre-selected for the presence of the full length episomal plasmid. Interestingly, it was found that the promoter in the 5′ untranslated region could be replaced with the CMV promoter without significantly affecting the retrotransposition frequency, and that the 3′ untranslated region could be completely deleted without any deleterious effect. When some of the integration sites of the L1/reporter element were cloned and the 5′ junctions sequenced, the elements were found to have been variably truncated 5′ of the selectable marker gene. This results in an integrated element that is presumably incapable of further retrotransposition, as: 1) the 5′ promoter is truncated, thus no mRNA intermediate would be transcribed in the forward orientation; 2) the essential ORF (at least ORF 1, and in some cases ORF 2 also) functions are deleted; and 3) even if the ORF 1 and ORF 2 gene products were to be provided in trans, it has been suggested that the retrotransposition process might be designed to ensure that only mRNA that is in cis with the ORFs is preferentially retrotransposed, perhaps by interaction of the nascent ORF 2 protein with the polyA tail of its own transcript during translation (Boeke, 1997).
Although use of retrotransposons as gene delivery vehicles has been previously suggested (Hodgson et al., 1997; Kingsman et al., 1995), and in fact retrotransposons such as rat VL30 elements have been found capable of being packaged and transmitted by MLV (Chakraborty et al., 1994; Torrent et al., 1994), thus far the
Higo Collin
Kasahara Noriyuki
Mitani Kohnosuke
Soifer Harris
Bingham & McCutchen LLP
Guzo David
University of Southern California
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