Chimeric viral packaging signal without gag gene sequences

Details Chimeric viral packaging signal without gag gene sequences Chimeric viral packaging signal without gag gene sequences

2000-09-18

2003-06-03

Housel, James (Department: 1648)

Chemistry: molecular biology and microbiology

Vector, per se

C424S185100, C424S187100, C424S199100, C435S069100, C435S456000, C514S04400A, C536S023100, C536S023400, C536S023720

Reexamination Certificate

active

06573091

ABSTRACT:

BACKGROUND OF THE INVENTION
The employment of retrovirus derived vectors in biotechnological applications has been standard practice for many years. For example, early retrovirus-derived vectors are described in Wei et al.,
J. Virol
., 39:935-44 (1980) and Shimotohno et al.,
Cell
, 26:67-77 (1981). Retroviruses are single-stranded RNA viruses. During an infection process in a host subject, such as a human, the RNA viruses are reverse transcribed into double stranded DNA. The double stranded DNA is subsequently integrated into the host cell DNA, and the virus becomes a permanent part of the host cell DNA. Once integrated, the virus is capable of expression of more viral RNA as well as the proteins that make up the virion. As retroviruses are usually not lytic, the retrovirus can continue to produce virus particles that bud from the surface of the cell.
Typically, modern retroviral vectoring systems consist of (1) RNA molecule(s) bearing cis-acting vector sequences needed for transcription, reverse-transcription, integration, translation and packaging of viral RNA into the viral particles, and (2) helper virus particles, budding from vector producer cells (VPCs), which express the trans-acting retroviral gene sequences (as proteins) needed for production of virus particles. By separating the cis- and trans-acting vector sequences completely, the virus is unable to maintain replication for more than one cycle of infection. The trans-acting vector sequences make empty virions (viral particles), whereas cis-acting vector sequences are capable of perpetuation or duration, only in the presence of the helper particles. Thus, cis-acting vector sequences and retroviral helper cells are the two essential components of modern retroviral vectoring systems.
Retrovirus-derived vectors (RVs) have been used in the majority of gene therapy clinical trials for a variety of reasons. For example, RVs can permanently integrate and express foreign genes, thus overcoming the problem of transient (short-term) expression, which is a significant problem of DNA transfection. Retroviruses, however, also suffer from several significant drawbacks. For example, retroviruses usually infect only dividing cells, and have a lower titer than some DNA viruses, such as adenovirus 5-derived vectors having titers of >10
11
transducing units/milliliter (TU)/ml. In addition, genetic recombination or “cross-over” can occur during replication (or at the DNA level), which can lead to outbreaks of replication competent retrovirus (“RCR”). RCRs occur as a result of regenerating the complete viral genome by genetic recombination. There are at least two different mechanisms by which this can occur. First, similar or identical overlapping nucleic acid sequences present on two separate DNA molecules (i.e., vector and helper sequences) can genetically recombine. Second, two separate RNA strands can serve as templates for cDNA synthesis during replication of the vector/virus, and genetic recombination can occur during DNA synthesis, leading to RCR. An additional confounding factor are the endogenous retroviral gene sequences that are present in the genome of the cells in which the virus is replicating. These retroviral gene sequences provide an additional source for generating RCR. Thus, it is important to separate the cis- and trans-acting sequences completely (i.e., no sequence overlap), and to provide a host cell genome that is devoid of closely related endogenous viral genes.
Unfortunately for most genetic therapies, RCR outbreaks have been detected in >15% of all lots of manufactured vectors tested prior to clinical trials. RCR are potentially lethal to primates and humans, and are therefore prohibited by the regulatory authorities. Significantly, the cost for detecting RCR can reach up to $100,000 per clinical batch. Often, however, RCR outbreaks often occur late in a vector production run as the VPCs are expanded. This suggests that a last minute outbreak might not be detected unless all of the clinical supernatant was tested. Thus, there is an ‘uncertainty principle’ that prevents complete assurance of catching the outbreak.
Still more unfortunately, many genetic therapy patients are immunecompromised (such as AIDS patients), and have reduced host defenses against oncogenic viruses, should an outbreak of RNA tumor virus occur. Clearly, this result presents many potential dangers. For example, an onco-retrovirus vector and the HIV retrovirus could infect the same cell. This mixed infection would most likely result in hybrid retrovirus particles containing both HIV-tropic and murine leukemia virus (“MLV”)-tropic particles (ie., the hybrid particles can share their host ranges, enabling a broader scope of infection). Such mixed pseudotype infections have the expected, expanded cellullar tropism (Lusso et al.,
Science
, 247:848-852 (1990)). MLV-tropisms include the so-called amphotropic (or 4070A strain) and gibbon ape leukemia virus (GALV)-tropic viruses, which are each capable of infecting a wide variety of human cells (as opposed to the primarily T-cell tropism of the native HIV-1 virus). Thus, a mixed virus infection could lead to HIV-1 infection of an expanded human cell repertoire, and possibly to a situation where the virus spreads like a common infection.
Of equal concern would be a genetic recombination event between MLV and HIV-1, wherein the HIV-1 envelope acquires a recombinant tropism, such as amphotropism or GALV-tropism. This could potentially provide a new HIV virus that could spread easily between humans, infecting most cells. It might be possible for only a small portion of the murine envelope glycoprotein gene to be present in order for this situation to occur (thus it might not disrupt other essential HIV-1 genes).
Therefore, a need exists for safe and efficient vectors for the transmission of genetic materials in a mammal. The genetic elements utilized in such vectors should be able to transmit genetic material that can be employed in gene therapy and cell therapy protocols as well as other biotechnological applications.
SUMMARY OF THE INVENTION
The present invention provides a chimeric viral packaging signal that can be employed in a vector for transmission of genetic material. The packaging signal contains an essential packaging nucleic acid sequence isolated from a mammalian type C retrovirus that is functionally joined to at least one non-essential packaging nucleic acid sequence that is isolated from a murine VL30 nucleic acid sequence. Additionally, the non-essential packaging nucleic acid sequence lacks a gag gene sequence.
Preferably, a murine leukemia virus is the mammalian type C retrovirus source for the essential packaging nucleic acid sequence. Additionally, the packaging signal can further contain at least one long terminal repeat nucleic acid sequence. The long terminal repeat nucleic acid sequence can be isolated from several possible sources, such as, a murine VL30 element, a retrovirus or a retrotransposon. Preferably, a packaging signal of the invention is employed in a retroviral vector.
The invention also provides a chimeric viral packaging signal that contains a long terminal repeat nucleic acid sequence isolated from a type C retrovirus or retrotransposon and operably linked to an essential packaging nucleic acid sequence. The essential packaging sequence is typically isolated from a mammalian type C leukemia virus and is operably linked to at least one non-essential packaging nucleic acid sequence. Preferably, the non-essential nucleic acid sequence is isolated from a murine VL30 nucleic acid sequence and lacks a gag gene sequence. Additionally, the prepared packaging signal is capable of packaging viral RNA or vector RNA into a retroviral capsid.
In one embodiment, the long terminal repeat nucleic acid sequence employed in a packaging signal of the invention is isolated from a murine retrovirus, a murine VL30 nucleic acid sequence, a retrotransposon, a simian retrovirus, an avian retrovirus, a feline retrovirus, a lentivirus, an avian retrovirus or a bovine retrovirus.

Affiliated with

Also associated with

Rating

Chimeric viral packaging signal without gag gene sequences does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Chimeric viral packaging signal without gag gene sequences, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Chimeric viral packaging signal without gag gene sequences will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3132629