Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-06-01
2003-12-02
Nguyen, Dave T. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S091400, C435S252300, C435S455000, C536S023100, C514S04400A, C424S093200
Reexamination Certificate
active
06656706
ABSTRACT:
I. TECHNICAL FIELD
The invention relates to nucleic acids comprising mutated HIV-1 gag/pol and SIV gag gene sequences which are capable of being expressed independently of any SIV or HIV regulatory factors. The invention also relates to nucleic acids comprising a mutated SIV env gene sequence, which is capable of being expressed independently of any SIV or HIV regulatory factors. The preferred nucleic acids of the invention are capable of producing infectious viral particles.
The invention also relates to vectors, vector systems and host cells comprising the mutated HIV-1 gag, HIV-1 pol, SIV gag and/or SIV env gene sequences. The invention also relates host cells comprising these nucleic acids and/or vectors or vector systems. The invention also relates to the use of these nucleic acids, vectors, vector systems and/or host cells for use in gene therapy or as vaccines.
II. BACKGROUND
Until recently, gene therapy protocols have often relied on vectors derived from retroviruses, such as murine leukemia virus (MLV). These vectors are useful because the genes they transduce are integrated into the genome of the target cells, a desirable feature for long-term expression. However, these retroviral vectors can only transduce dividing cells, which limits their use for in vivo gene transfer in nonproliferating cells, such as hepatocytes, myofibers, hematopoietic stem cells, and neurons.
Lentiviruses are a type of retrovirus that can infect both dividing and nondividing cells. They have proven extremely efficient at providing long-term gene expression (for up to 6 months) in a variety of nondividing cells (such as, neurons and macrophages) in animal models. See, e.g., Amado et al., Science 285:674-676 (July 1999). It has been proposed that the optimal gene transfer system would include a vector based on HIV, or other lentivirus, that can integrate into the genome of nonproliferating cells. Because retroviruses integrate in the genome of the target cells, repeated transduction is unnecessary. Therefore, in contrast to an adenoviral vector capable of in vivo gene delivery, problems linked to the humoral response to injected viral antigens can be avoided. See, e.g., Naldini et al., Science, 272:263-267 (1996), p. 263.
HIV and other lentiviruses have a complex genome that, in addition to the essential structural genes (env, gag, and pol), contains regulatory (tat and rev) and accessory genes (vpr, vif vpu, and nef). HIV has evolved to efficiently infect and express its genes in human cells, and is able to infect nondividing cells such as macrophages because its preintegration complex can traverse the intact membrane of the nucleus in the target cell. This complex contains, in addition to the viral DNA, the enzyme integrase, the product of the vpr gene, and a protein encoded by the gag gene called matrix. The matrix protein enables the preintegration complex to pass into the nucleus to access the host DNA. Lentiviruses cannot efficiently transduce truly quiescent cells (cells in the G
0
state). However, unlike murine retroviral vectors, in addition to being able to infect dividing cells, HIV-based vectors can achieve effective and sustained transduction and expression of therapeutic genes in nondividing cells, such as hematopoietic stem cells and in terminally differentiated cells such as neurons, retinal photoreceptors, muscle, and liver cells. See, e.g., Amado et al. (July 1999) and Klimatcheva et al., Frontiers in Bioscience 4:d481-496 (June 1999), and the references cited therein.
Although lentiviral vectors can be efficient gene delivery vehicles, there are safety concerns due to their origin. Therefore, the field has turned its attention to the development of vectors and production systems with built-in safety features to prevent the emergence of replication competent lentivirus (RCL). For example, in most laboratory applications, lentiviral vectors are generally created in a transient system in which a cell line is transfected with three separate constructs: a packaging construct, a transfer construct, and an envelope encoding construct. The packaging construct contains the elements necessary for vector packaging (except for env) and the enzymes required to generate vector particles. The transfer construct contains genetic cis-acting sequences necessary for the vector to infect the target cell and for transfer of the therapeutic (or reporter) gene. The lentivirus env gene is generally deleted from the packaging construct and instead the envelope gene of a different virus is supplied in a third vector “the env-coding vector”, although the lentiviruses env gene may be used if it is desired that the vector be intended to infect CD4
+
T cells. A commonly used envelope gene is that encoding the G glycoprotein of the vesicular stomatitis virus (VSV-G), which can infect a wide variety of cells and in addition confers stability to the particle and permits the vector to be concentrated to high titers (see, e.g., Naldini et al., Science 272:263-267 (1996) and Akkina et al. J. Virol. 70:2581 (1996). The use of three separate constructs and the absence of overlapping sequences between them minimizes the possibility of recombination during lentivirus (transfer) vector production. In addition, because no viral proteins are expressed by the lentiviral (transfer) vector itself, they do not trigger an effective immune response against cells expressing vector in animal models (a particular problem with vectors based on adenovirus). See, e.g., Amado et al., Science 285:674-676 (July 1999) and the references cited therein. See also Naldini et al. Science 272:263-267 (1996).
The initial packaging plasmids contained most HIV genes except for env. In an effort to improve safety, subsequent HIV vectors have been produced in which the packaging plasmid is devoid of all accessory genes. This process does not interfere with efficient vector production and significantly increases the safety of the system because potential RCLs lack the accessory genes necessary for efficient replication of HIV in humans. Although these vectors can transduce growth-arrested cell lines and neurons in vivo, they have been reported to not efficiently transduce macrophages. The accessory gene vpr is believed to be necessary for HIV infection of these cells using these HIV vectors. See, Zufferey et al., Nature Biotechnol. 15:871-875 (1997). In contrast, as discussed later herein, the HIV-based lentiviral vectors of the present invention do not need any HIV accessory genes in order to be able to infect human macrophages and the other cells tested.
The requirement of vpr or vif for efficient transduction of liver cells has also been reported. See, e.g., Kafri et al., Nature Genet. 17:314 (1997). These results indicate that the requirement of accessory genes for efficient lentivirus-mediated gene transfer is dependent on the type of cell chosen as target, suggesting that future applications of lentiviral vectors may involve vector constructs with different accessory genes, as needed.
Zufferey et al., (1997) describe an HIV vector system in which the virulence genes, env, vif, vpr, vpu, and nef have been deleted. This multiply attenuated vector conserved the ability to transduce growth-arrested cells and monocyte-derived macrophages in culture, and could efficiently deliver genes in vivo into adult neurons. The packaging plasmids described Zufferey et al. (1 997) and Naldini et al. (1996) encode Rev and Tat, in addition to Gag and Pol.
Lentiviral vectors engineered to become packaged into virions in the absence of the regulatory gene tat have also been described. See, e.g., Kim et al., J. Virol. 72:811-816 (1998) and Miyoshi et al. J. Virol. 72:8150-8157 (1998). In these vectors the tat gene has been removed from the packaging plasmid. Kim et al. state that tat is not necessary as long as the serial 5′ LTR promoter is replaced with a strong constitutive promoter. It also has other advantages for HIV therapy. Replacement of the HIV-1 LTR with a constitutive HCMV promoter permits the use of anti-Tat molecules such as Tat transdom
Nguyen Dave T.
The United States of America as represented by the Department of
Townsend and Townsend / and Crew LLP
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