CZ2003784A3 - Enhanced conditionally replicating vectors, processes of their preparation and their use - Google Patents

Abstract

The present invention provides improved conditionally replicating vectors that have improved safety against the generation of replication competent vectors or virus. Also disclosed are methods of making, propagating and selectively packaging, modifying, and using such vectors. Included are improved helper constructs, host cells, for use with the improved vectors as well as pharmaceutical compositions and host cells comprising the vectors, the use of vector containing host cells to screen drugs, and methods of using the vectors to determine gene function. The methods also include the prophylatic and therapeutic treatment of disease, especially viral infection, and HIV infection in particular.

Description

The present invention provides improved conditionally replicating vectors, methods of making, propagating and selectively enveloping, modifying and using conditionally replicating vectors and lentiviral vectors. Significantly, these vectors have a reduced ability to become replication competent and thus exhibit increased safety in use.

Also provided are certain nucleotide and amino acid sequences relevant to such vectors, pharmaceutical compositions, and host cells comprising the vectors, use of these host cells for drug screening, and methods of using such vectors to determine gene function. These methods also include the prophylactic and therapeutic treatment of diseases, particularly viral infections and especially HIV infections. The present invention is also directed to direct and indirect methods of making new vaccines for the treatment of infectious diseases, cancer and other diseases of genetic nature and for diagnostic purposes. Other methods and compositions of the invention include the use of conditionally replicating vectors and lentiviral vectors in gene therapy and other applications.

BACKGROUND OF THE INVENTION

The discovery of human immunodeficiency virus (HIV), lentivirus, as the cause of acquired immune deficiency syndrome (AIDS), has turned research into the mechanisms underlying the viral infection cycle and the pathogenesis of viruses. Studies of these mechanisms have been provided by researchers with an increasing number of targets for the development of antiviral agents, effective not only against HIV but also against HIV products, its genome and also against other viruses. These antiviral agents, especially those directed against HIV, can be divided into four categories, depending on their mode of action. These categories include reverse transcriptase inhibitors, viral cell entry competitors, vaccines, and protease inhibitors, as well as the more modern categories referred to herein as "genetic antiviral agents".

Each type of antiviral agent generally has its own, associated, beneficial properties and limitations, and must be judged from the requirements in a particular therapeutic situation. Antiviral agents such as zidovudine (3'-azido-3'-deoxythymidine, also known as AZT), protease inhibitors and them.

similarly, they can be delivered to the cells of the patient's body relatively easily and have been extensively studied. Targeting one specific factor in the viral infection cycle has been found to be relatively ineffective against HIV. The primary cause of this is that HIV strains change rapidly and become resistant to agents that have only one site of action (Richman, AIDS Res. And Hum. Retrovir., 8, 1065-1071 (1992)). Accordingly, the problems of genetic variability and rapid mutation of the HIV genome have forced into consideration new antiviral strategies for the treatment of HIV infections. Among these options, antiviral agents are attractive because they act at many different levels of intracellularly.

Genetic antiviral agents differ from other therapeutic agents in that they are transferred as molecular elements to target cells where they protect these cells from viral infection (Baltimore, Nature, 325, 395-396 (1988) and Dropulič et al., Hum. Gene Ther., 1994, 5, 927-939). Genetic antiviral agents may have any genetic sequence and include, but are not limited to, antisense molecules, RNA banners, transdominant mutants, interferons, toxins, immunogens, and ribozymes. In particular, ribozymes are genetic antiviral agents of the antisense type that cleave target RNAs, including HIV RNA, in a sequence-specific manner. The specificity of ribozyme-mediated cleavage of the target RNA suggests the possible use of robiobymes as therapeutic inhibitors of viral replication, including HIV replication. Various types of ribozymes such as "hammerhead" and "hairpin" may be used in anti-HIV strategies (see, eg, US Patent Nos. 5,144,019; 5,180,818 and 5,272,262 and PCT Patent Application Nos. WO 94 / 01 549 and WO 93/23 569). Both the hammerhead and hairpin ribozymes can be engineered to cleave any target RNA that contains the GUC sequence (Haseloff et al., Nature, 334, 585-291 (1988); Uhlenbeck, Nature, 334, 585 (1987)) Hampel et al., Nuc. Acids Res., 18,299-304 (1990) and Symons, Ann. Rev. Biochem., 61, 341-371 (1992)). Generally speaking, hammerhead ribozymes have two types of functional domains, a conserved catalytic domain, surrounded by two hybridization domains. These hybridization domains bind to sequences that surround the GUC sequence and the catalytic domain cleaves the target RNA from the 3 'end of the GUC sequence (Uhlenbeck (1987) supra; Haseloff et al., (1988) supra and Symons (1992) supra).

Numerous studies have confirmed that ribozymes may be at least partially effective in inhibiting HIV propagation in tissue culture cells (see, Sarver et al., Science, 247, 1222-1225 (1990)); Sarver et al., NIH Res., 5.63-67 (1993a); Dropulic et al., J. Virol., 66, 1432-141 (1992); Dropulic et al., Methods: Comp. Meth. Enzymol., 5,43-49 (1993); Ojwang et al., PNAS 889: 1080-10806 (1992); Yu et al., PNAS, 90, 6340-6344 (1993) and Weerasinghe et al., J. Virol., 68, 5531-5534 (1991). In particular, Sarver et al., ((1990) supra), has shown that hammerhead ribozymes designed for cleavage within the transcribed region of the HIV gag gene, i.e.,

anti-gag ribozymes, can specifically cleave HIV gag RNA in vitro. In addition, when cell lines expressing anti-gag ribozymes are contacted with HIV 1, 50-100 fold inhibition of HIV replication was observed. Similarly, Weerasingh et al. ((1995) supra) that retroviral vectors encoding ribozymes engineered for cleavage within the U5 sequence of HIV 1 RNA are responsible for HIV resistance to transduced cells upon subsequent contact with HIV. Although different clones of transduced cells demonstrate different levels of resistance to attack, as determined by the promoter system used to control ribozyme expression, most ribozyme secreting cell lines have undergone HIV expression after some limited time in culture.

Transducing tissue culture cells with provirus into a nef gene (which is essential for viral replication in tissue cultures) into which a ribozyme has been transduced and whose hybridization domains were specific for the U5 region of HIV, have been shown to inhibit viral replication in transduced cells 100-fold compared to cells transduced with wild-type provirus (see, eg, Dropulič et al. (1992) and (1993), supra). Hairpin ribozymes have similarly been found to inhibit HIV replication in T cells transduced with vectors containing U5 hairpin ribozymes and infected with HIV (Ojwang et al. (1992, supra). Other studies have shown that vectors containing ribozymes, expressed by tRNA promoters, also inhibit various HIV strains (Yu et al. (1993) supra).

The transfer of ribozymes or other genetic agents to the cellular targets of HIV infection (eg, CD4 + T cells and monocytic macrophages) was a major impediment to effective genetic therapeutic treatment of AIDS. Current approaches to targeting cells of the hematopoietic system (i.e., the primary targets of HIV infection) call for the introduction of therapeutic genes into precursor multipotent stem cells, which, upon differentiation, mature into T-cells or possibly into mature CD4 + T-cells themselves. However, stem cell targeting is problematic since these cells are difficult to cultivate and transduce in vitro. Targeting circulating T lymphocytes is also problematic because these cells are so widely scattered that it is difficult to reach all target cells using vector delivery systems. In addition, macrophages should be considered as cellular targets as they are the main reservoir for the spread of the virus to other organs. However, since the macrophages are no longer differentiated and therefore no longer subject to cell division, they are not readily transduced by commonly used vectors.

Accordingly, the currently prevalent approach to HIV treatment uses replication deficient viral vectors and helper cell lines (see, eg, Buchschacher, JAM A, 269 (22), 2880-2886 (1993); Anderson, Science, 256: 808-813 (1992); Miller, Nature, 357: 455-460 (1992); Mulligan, Science, 260: 926-931 (1993); Friedmann, Science, 244, 1275; To 1281 (1989) and Coumoyer et al., Ann. Rev. Immunol., 11, 297-329 (1993)) for the introduction of foreign genes into cells susceptible to viral infection (such as HIV infection) that specifically interfere with virus replication or that cause the death of the infected cell (Buchschacher (1993), supra). Such replication deficient viral vectors contain, in addition to the foreign gene of interest, a cis acting sequence necessary for viral replication, but not sequences that encode essential viral proteins. As a result, such a vector is unable to complete the viral replication cycle and the helper cell line that contains and constitutively expresses the viral genes in its genome is used to propagate it. Upon introduction of a replication-defective viral vector into a helper cell line, the proteins necessary for viral particle formation are provided in vector and the vector viral particles capable of infecting target cells and expressing a gene that interferes with virus replication or causes death of infected cells are produced. virus.

Such replication deficient retro viral vectors include adenoviruses and adeno-associated viruses, as well as those viral vectors used in clinical trials for the treatment of HIV, and in particular a mouse amphotropic retroviral vector known as Moloney Mouse Leukemia Virus (MuLV). These defective viral vectors are used to transduce CD4 + cells with genetic antiviral agents such as anti-HIV ribozyme, with varying degrees of success (Sarver et al. (1990) supra); Weerasinghe et al. (1991) supra; Dropuli et al. (1993) supra; Ojwang et al. (1992), supra and Yu et al. (1993) supra). However, these vectors are greatly limited for use in HIV gene therapy. For example, the high frequency of transduction is particularly important in HIV treatment, where the vector should transducate either rare CD34 + ancestors of hematopoietic stem cells or widespread target CD4 + T-cells, most of which are already infected with HIV during the clinical "latent" stage of the disease. However, MuLV vectors are difficult to obtain at a higher titer, thus causing a slight transduction. In addition, long-term expression of transduced DNA was not obtained in predecessors of CD34 + stem cells, mainly after differentiation into mature lymphocytes. In addition, the use of defective viral vectors requires ex vivo gene transfer strategies (see, eg, US Patent No. 5,399,346), which can be expensive and beyond the capabilities of the general population.

These drawbacks, coupled with the use of commonly available vectors for the genetic therapeutic treatment of AIDS, have led researchers to find new viral vectors. One such vector is HIV itself. HIV vectors have been used for infectious studies (Page et al., J. Virol., 64, 5270-5276 (1990)) and for the introduction of genes (as well as suicide genes) into CD4 + cells, mainly into CD4 + HIV-infected cells (see e.g., Buchschacher et al., Hum. Gener. Ther., 3, 391-397 (1992); Richardson et al., J. Virol., 67, 3197-4005 (1993); Carroll et al.

J. Virol., 68, 6047-6051 (1994) and Parolin et al., J. Virol., 68: 3888-3895 (1994). The strategy of these studies is to use HIV vectors to introduce genes into CD4 + T cells and monocytic cells.

At present these vectors are extremely complex. In addition, the use of these vectors is associated with the risk of developing wild-type HIV via intracellular recombination. As a result of co-transfection / coinfection of defective vector sequences and helper virus, recombination between homologous regions of the viral genome was observed (Inoue et al., PNAS,

88, 2278-282 (1991)). The observed in vitro complementation suggests that a similar replication-defective HIV vector could recombine in vivo, thereby exacerbating an existing HIV infection. The fact that retro viruses encapsulate two RNA genomes into one virion has led researchers to believe that retro viruses carry two viral RNAs to overcome any genetic defects caused by complementation and / or recombination (Inoue et al. (1991), supra).

In addition to the risk of intracellular recombination resulting in the emergence of wild-type HIV, HIV vectors also carry the inherent risk of mutation in vivo, which increases the pathogenicity of this viral vector. This led to Sarver et al. (AIDS Res. And Hum. Retrovir., 9,483-487 (1993b)) for speculation regarding the development of second generation recombinant HIV vectors that are replication competent but no longer pathogenic. Such vectors, in comparison to the predominantly used non-replicating vectors (i.e., replication defective vectors), continue to replicate in the patient and thus undergo constant competition with wild-type HIV. However, such vectors are not available until now.

Ideally, the best treatment option for an infected individual would occur at the time of inoculation before the virus infects the host. However, this is difficult to achieve because many individuals do not realize that they have been infected with HIV, to the clinically latent phase of the disease. On this basis, the stage at which the most urgent need for antiviral intervention is is the course of clinical latency. Therapy at this stage requires that the challenge represented by a large number of already infected CD4 + lymphocytes that contain viral genomes. This is not a simple task, as is clear from the fact that to date HIV remains incurable and is currently available therapies only partially curable. The active vaccine is not approaching, and although it has been shown that reverse transcriptase and protease inhibitors prevent HIV replication in tissue cultures, the development of viral resistance in vivo has led to the failure of this treatment. HIV gene therapy has so far been of little benefit to the vast number of HIV-infected individuals, which are over 30 million to date.

In light of the above, it is increasingly important to develop long-term and sustained immunological responses to certain pathogens, particularly viruses, particularly in the context of

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with AIDS and cancer. Live-attenuated (LA) vaccines using replication-competent but non-pathogenic viruses are considered (Daniel et al., Science, 258: 1938-1941 (1992) and Desrosiers, AIDS Res. & Human Retrovir., 10, 331-322 (1994)). However, such non-pathogenic viruses, which differ from the corresponding wild-type virus deletions in one or more genes, either (i) cannot elicit a protective immune response because the antigen is not persistent (because the LA virus is not effectively replicating) or (ii) LA the virus replicates, but has a different pathogenic potential, as evidenced by the ability of this LA virus to cause disease in animal models of babies (Baba et al., Science, 267, 1823-1825 (1995)).

For the above reasons, there is still a need for alternative prophylactic and therapeutic treatments for viral infection, particularly in the context of AIDS and cancer. The present invention offers such alternative methods by providing a conditionally replicating vector. The invention also provides additionally the methods by which this vector can be utilized. Further provided by the present invention are helper vector constructs which complement the conditionally replicating vector by guaranteeing its replication and envelope as viral particles and virions. Various embodiments of such helper vector constructs are described. These and other objects and advantages of the present invention, as well as other features of the invention, will be apparent from the description of the invention which follows.

SUMMARY OF THE INVENTION

The present invention provides improved, conditionally replicating vectors, as well as improved means and methods for making and using said vectors. The conditionally replicating vector is characterized by the ability to replicate only in the host cell that permits replication of the vector. These improved vectors thus provide increased security by reducing the risk of re-sharing. As such, these vectors may also be referred to as "reduced recombination" vectors.

In one aspect of the invention, the improved conditionally replicating vector comprises at least one nucleic acid sequence whose presence and transcription or translation confer upon the vector in replication allowing the host cell a selective advantage over the wild-type virus strain corresponding to the virus from which the vector was derived. The improved, conditionally replicating vector preferably comprises at least one nucleic acid sequence that confers a selective advantage on the vector in replication over any other competing genomes or genomic elements. The genomic element referred to herein is defined as a nucleic acid sequence in a host cell that is not derived from this vector or from any wild-type virus and that can compete, interfere, or affect vector replication in the host cell. host cell. This genomic element is also not a complete genome, such as the host cell genome or other virus or vector. The selective advantage in replication is conferred by the presence, transcription or translation of said nucleic acid sequence.

In another preferred embodiment, the conditionally replicating vector is a retro virus, particularly a lentivirus, and comprises at least one nucleic acid sequence whose presence, transcription or translation confer a selective advantage over a cell infected with the wild-type virus strain corresponding to the host cell infected with the vector. the virus from which this vector was derived. Alternatively, this vector is not entirely derived from the wild-type strain, but is a chimeric containing components derived from more than one wild-type virus. The more than one wild-type virus may be different (or heterologous) viruses or other strains or isolates of a single virus. Preferably, said nucleic acid sequence can be expressed, thereby achieving a prophylactic effect in the host cell. These results are a persistent benefit to the cell because this nucleic acid sequence prevents any infectious virus from replicating at a level that causes the cell to die.

Another preferred embodiment of the present invention is an improved, conditionally replicating plasmid vector comprising at least one nucleic acid sequence that confers a selective advantage on the vector in replication over any other competing vector or plasmid molecule. For example, such a vector may comprise a sequence (e.g., a ribozyme or antisense sequence) capable of cleaving or degrading or resulting in cleavage or destruction resulting in cleavage of a competing vector or plasmid when placed in the vector. If both the competing vector and the helper need to be decomposed, the vector of the invention is designed to contain other sequences that give it an overall selective advantage for replication. For example, a vector of the invention may contain an atisense sequence that disrupts both the competing vector and the helper, but would have a selective advantage for replication since it additionally contains a second first nucleotide sequence (e.g., a promoter that produces more vector RNA than the competing vector RNA) the copy of this vector in the transduced cell is higher than in the competing genome, or the packaging signal that is present in the vector but not in the helper), which provides a conditionally replicating vector with an overall selective advantage. Thus, at least the first nucleotide sequence further provides, in this embodiment, the complex selective advantage of replicating the vector of the invention, since the two first nucleotide sequences (i.e., the first sequence with the first nucleotide target the competing vector and / or helper) and the second sequence with the first nucleotide provides a selective advantage for replication ) act in concert to achieve a selective selective effect. Synergic ·· ··· · «· 9999

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Thus, the effects of multiple first nucleotide sequences may lead to enhanced selection conditions for the conditionally replicating vector, wherein any single first nucleotide sequence allows for discriminatory selection against the competing genome.

Cleavage or disruption of the competing vector or plasmid also reduces the possibility of full-length recombination in the vector, so that the vector then has a "reduced recombination" ability. The vector can be protected from cleavage by digestion of the sequence so that it cannot be targeted by a ribozyme or natisense sequence. The vector may further be protected from digestion by a genetic modification or genomic version of the vector, causing the RNA to be otherwise aligned so that the genomic vector RNA is not significantly cleaved by the competing vector or helper. Alternatively, the helper may be similarly and uniquely genetically engineered, for example by inserting splicing elements to introduce the helper into the splicing complex, in constructing the vector genomic RNA to contain the first nucleic acid sequences that are present only in the uncut and not the spliced vector RNA. These non-limiting examples of improved vectors are particularly advantageous when used in the production of a viral vector or plasmid vector and in cloning or subcloning nucleic acid sequences in such vectors, as well as in their use in mutagenesis, expression of inducible genes and the like.

Also provided by the present invention is a pharmaceutical composition comprising a conditionally replicating vector of the invention and a pharmaceutically acceptable carrier. In addition, a host cell comprising a conditionally replicating viral vector is provided. Also included is a vector, which vector, if DNA is, comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2, 3,4,5,6,14, wherein at least one N is mutated, 15 and 16, and wherein said vector, if RNA, comprises a nucleotide sequence encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2, 3, 4, 5, 6, 14, wherein at least one N is mutated, and 15 and 16, and the isolated and purified nucleic acid molecules described herein. Similarly, methods for genetically engineering a vector with a ribozyme, a method of modifying a vector, and a method of propagating and selectively enveloping a conditionally replicating vector without using a enveloping cell line are provided.

In yet another aspect of the invention there is provided a method of therapeutic and prophylactic treatment of a host cell from a viral infection. In particularly preferred embodiments, the treatment is influenced in said host cell by the dominant phenotype supplied by it which inhibits viral infection, or by delivery of a phenotype that inhibits infection by other viruses. It is preferred that these other inhibited viruses include a wide variety of viral strains. In addition, these methods may include the use of an auxiliary "helper" expression vector. vector 'or' helper vector construct ', cytotoxic drug, protein / factor or protease inhibitor / reverse transcriptase. For example, the method may be used to inhibit viral replication, to treat diseases (including cancer, genetic, infectious, vascular and other diseases), to perform efficient gene transfer ex vivo and in vivo, to provide safe vector production, to determine gene function, or to expressing the gene of interest in the host cell.

In yet another aspect of the invention, there is provided a method of using a host cell comprising a conditionally replicating vector of the invention for detecting drug / factor-protein interaction. This method allows the characterization of a protein and the screening of drugs, factors or other proteins for activity or physical interaction with respect to a given protein encoded by this vector. This method further allows identification and characterization of proteins that are operably linked to a given protein encoded by the vector. For example, if the encoded protein is a transcription factor, its expression by a vector of the invention allows the identification of genes regulated by this transcription factor.

The present invention also provides compositions and conditions for storing vectors prior to use under various conditions. Examples of such storage include storage at -80 ° C, -20 ° C and 4 ° C in the presence of various carriers.

Other embodiments of the invention include generic lentiviral vectors, modifications to helper vector constructs that serve to reduce, minimize, or eliminate recombination of a helper vector with a conditionally replicating vector to form a replication competent vector or virus (RCV). Therefore, the present invention also encompasses helper vectors that serve to prepare "reduced recombination" vectors. The helper vector of the invention also advantageously increases the titer of the conditionally replicating vector produced to a level greater than 10 ⁷ transduction units per milliliter. Other embodiments include vector concentration using high speed (but not ultra) centrifuges and chromatographic, ultrafiltration and diafiltration methods of concentration and purification.

Modification of the helper vector of the invention includes insertion of a ribozyme, such as an anti-U5 ribozyme, or antisense sequence that cleaves or results in destruction or inactivation of a conditionally replicating vector when the helper vector and the conditionally replicating vector are located together or together packaged. In one embodiment, the helper components are completely integrated on a single plasmid construct, resulting in a two-plasmid functional helper-vector system that effectively produces unconcentrated vector supernatant titers greater than 10 ⁷ transduction units per ml (see Figure 3). In another embodiment, the nucleotide sequence of the helper vector is degenerate to be

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minimized recombination with the conditionally replicating vector. In yet another embodiment, the helper vector comprises heterologous trans-acting elements for use in packaging a conditionally replicating vector. Examples of such elements include, but are not limited to, VSV-G vesicular stomatitis virus envelope protein, RDI 14 envelope protein, rabies virus envelope protein, Gibbon Leukemia Virus (GAL V) envelope protein, and chimeric envelope proteins. Other modifications also include heterologous, Rev Response Elements (RRE), Posttranscriptional Control Elements (PRE), or Constitutive Transport Elements (CTE).

In yet another embodiment, the helper vector construct comprises a splice donor, splice acceptor sites, or sites for degenerate or humanized nucleotide sequences. For example, splice sites may be located such that the enveloping signal and / or the RRE can be removed from the transcribed RNA splicing. In addition, intron insertion sites are present in vector or helper constructs such that dimerization, co-localization or recombination between the conditionally replicating vector and the helper vector or other competing genome or genomic element is minimized. Intron insertions can be directed to result in the delivery of helper RNA to splice complexes and away from the vector RNA. Surprisingly, some helper vector constructs have been found to increase the titer of a conditionally replicating vector.

The present invention provides improved conditionally replicating vectors, lentiviral vectors, and uses thereof. In addition to being selectively replicated, these vectors contain specific modifications to reduce the likelihood of recombination as a result of the emerging replication of competence. Included are methods of inhibiting replication of a wild-type virus strain and methods for delivering a gene into cells, including such improved vectors. The method comprises contacting a host capable of being infected, at risk of infection, or preferably actually infected with a wild-type strain of virus, with an improved vector that is propagated only in a host that allows replication of the vector (i.e., non-pathogenic or non-pathogenic) disease, conditionally replicating vector (s)).

As described hereinbelow, the particular aim of the method is to induce a competitive infection in the host by such a non-pathogenic, conditionally replicating virus. Generally, a conditionally replicating vector of the invention comprises at least one nucleic acid sequence that provides a selective advantage for replication and extension to a conditionally replicating vector over a wild-type virus, and / or at least one nucleic acid sequence that provides a selective advantage for viral propagation particles into a host cell containing a conditionally replicating vector as compared to a host cell containing a wild-type virus.

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In a preferred embodiment of the invention, the vector comprises an HIV sequence and is used to treat HIV infection. The vector or host cell comprising the vector comprises at least one nucleic acid sequence that (1) provides a crHIV genome with a selective advantage over the wild-type HIV genome in enveloping progeny virions (i.e., cells in which they are both established) and / or (2) provides a host cell that produces a conditionally replicating vector (virus) with a selective advantage in producing a crHIV virion as compared to a host cell producing a wild-type virus. One method (but not limited to this invention) is to confer a selective advantage on the packaging of crHIV genomes by providing one or more ribozymes capable of cleaving wild type HIV genomes.

In another aspect of the invention, the vectors are provided with a reduced likelihood of recombination with wild-type constructs and helper constructs to obtain replication competent vectors. In addition, helper vectors, cells and packaging systems that also contribute to reduce recombination are also provided to further reduce the possibility of productive recombination to ensure that these vectors no longer conditionally replicate.

Wild virus types

According to the invention, a "virus" is an infectious agent consisting of a protein and a nucleic acid and which uses the genetic mechanisms of a host cell to produce viral nucleic acid-determined products. "Nucleic acid" means a DNA or RNA polymer that is single- or double-stranded, linear or circular and optionally contains synthetic, non-natural or modified nucleotides that are capable of incorporation into DNA or RNA polymers. The DNA polynucleotide preferably comprises genomic or cDNA sequences.

A "wild-type strain of virus" is a strain that does not contain any man-made mutations as described herein, ie, any virus that can be isolated in nature. Alternatively, the wild-type strain is any virus that has been cultivated in the laboratory, but still, in the absence of any other virus, is capable of producing genomes or virions of progeny as viruses that have been isolated in nature. For example, the molecular clone pNL4-3 HIV-1 described in the following examples is a wild-type strain available in the AIDS Research and Reference Reagent Program Catalog from the National Institutes of Health (see also Adachi et al., J. Virol., 59, 284). to 291 (1986)). pPSXB is an HIV-2 molecular clone, kindly provided by Dr Suresh Arya of the National Institutes of Health, Bethesda, Maryland, and described by Arya et al. Human immunodeficiency virus type 2 lentivirus vectors for gene transfer: expression and potential for helper virus-packaging. Hum Gene Ther. 1998 Jun 10; 9 (9): 1371-1380.

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In general, the method of the invention is preferably used for the treatment of viral diseases caused by viral infection. Desirably, the virus (as well as the vector, as discussed below) is an RNA virus, but may also be a DNA virus. RNA viruses are a diverse group that infects prokaryotes (eg, bacteriophages), as well as many eukaryotes, including mammals and in particular humans. Most RNA viruses have single stranded RNA as their genetic material, although at least one group has double stranded RNA as their genetic material. RNA viruses are classified into three main groups: positive-stranded viruses (i.e., those whose genome upon translation is translated into a protein and whose deproteinated nucleic acid is sufficient to induce infection), negative-stranded viruses (i.e. those whose genome, The virus transmitted is complementary to the meaning of the message and must be transcribed before translation can occur by virion-associated enzymes) and double-stranded RNA viruses. The method of the invention is preferably used for positive-stranded viruses, negative-stranded viruses and double-stranded RNA viruses.

RNA virus is used herein to mean Sindbis-like viruses (eg Togaviridae, Bromovirus, Cucumovirus, Tobamovirus, Ilarvirus, Tobravirus and Potexvirus), Picomavirus-like viruses (eg Picomaviridae, Caliciviridae, Comovirus, Nepovirus and Potyvirus), minus viruses chain (e.g., Paramyxoviridae, Rhabdoviridae, Orthomyxoviridae, Bunyaviridae and Arenaviridae), double-stranded viruses (e.g., Reoviridae and Bimaviridae), Flavivirus-like viruses (e.g., Flaviviridae and Pestivirus), Retrovirus-like viruses (e.g., Retrovirusidae), e.g. including, but not limited to, Nodaviridae.

A preferred RNA virus according to the invention is a Flaviridae virus, preferably a Filovirus virus, and in particular a Marburg or Ebola virus. Preferably, the Flaviviridae virus is a Flavivirus virus such as yellow fever virus, denge virus, West Nile virus, St. Petersburg virus. Louis encephalitis, Japanese encephalitis virus, Murray Valley encephalitis virus, Rocio virus, tick-borne encephalitis virus and the like.

Also preferred are Picomaviridae, preferably hepatitis A virus (HAV), hepatitis B virus (HBV) or non-A or non-B hepatitis virus.

Another preferred RNA virus is a virus of the family Retroviridae (i.e. retrovirus), in particular a virus of the genus or subfamily Oncovirinae, Spumavirinae, Spumavirus, Lentivirinae and Lentivirus. The RNA virus of the subfamily Oncovirinae is suitably a human T-lymphotropic virus type 1 or 2 (ie HTVL-1 or HTVL-2) or bovine leukemia virus (BLV), avian leukosis-sarcoma virus (eg Rous sarcoma virus (RSV), avian virus). myeloblastosis (AMV), avian erythroblastosis virus (AEV), Rous virus associated virus (RAV; RAV-0 to RAV-50), mammalian C-type virus (e.g., Moloney mouse leukemia virus (MuLV), Harvey mouse sarcoma virus ( HaMSV), Abelson's mouse leukemia virus (A-MuLV), AKR-MuLV, fox leukemia virus (FeLV), monkey sarcoma virus, reticuloendotheliosis virus (REV), spleen necrosis virus (SNV)), B-type virus (e.g., virus) mammary gland tumor in mice (MMTV)) and D-type virus (eg Mason-Pfizer simian virus (MPMV) and SAIDS viruses) Suitable human subfamily Lentivirus is human immunodeficiency virus type 1 or 2 (ie HIV-1 or HIV- 2), where HIV-1 was formerly referred to as Lymphoadenopathy Associated Virus 3 (HTLV-III) and Acquired Immune Syndrome nodeficiency virus (AIDS) associated virus (ARV)) or another HIV-1 or HIV-2 associated virus that has been identified and associated with AIDS or AIDS-like diseases. The abbreviation "HIV" or the term "AIDS virus" or "human immunodeficiency virus" is used herein to refer generally to these HIV viruses and to HIV-related or HIV-associated viruses. The other RNA virus of the subfamily Lentivirus is preferably Visna / maedi virus (eg, one that infects sheep), fox immunodeficiency virus (FIV), bovine lentivirus, monkey immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), and goat arthritis virus. -encephalitis (CAEV).

The virus according to the invention is also preferably a DNA virus. Preferably, the DNA virus is Epstein-Barr virus, adenovirus, herpes simplex virus, papillomavirus, vaccinia virus and the like.

Many of these viruses are classified as pathogens of the "Biosafety Level 4" (World Health Organization "Risk Group 4"), for which the maximum use of enclosed equipment applies in all laboratory work. However, properly trained personnel are aware of this and are able to take the precautions necessary to work with these viruses.

The "host cell" can be any cell, preferably a eukaryotic cell. Desirably, the cell is a lymphocyte (such as a T cell) or a macrophage (such as a monocytic macrophage) or a precursor of any of these cells, such as a hematopoietic stem cell. Preferably, these cells contain a CD4 + glycoprotein on their surface, i.e., they are CD4 +. However, it is desirable that the CD4 + T cell that has been infected with the AIDS virus has not yet become active (i.e., preferably did not express nef or more preferably, the expression of the CD4 + gene was not attenuated as discussed below). Moreover, the host cell is preferably a cell which lacks a CD4 marker and yet is able to be infected with the virus of the invention. Such a cell includes, but is not limited to, astrocyte, skin fibroblast, intestinal epithelial cell, endothelial cell, epithelial cell, dendrite cell, Langerhans cells, monocyte, hematopoietic stem cell, embryonic stem cell, sperm cell or egg cell, tree cell, mucosal cell, and the like. Preferably, the host cell is a cell of a eukaryotic, multicellular species (e.g., as opposed to a unicellular yeast cell), and more preferably, the cell is a mammalian, e.g., human cell.

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The cell may be present as a single entity or may be part of a larger set of cells. Such a "larger set of cells" may include, for example, cell culture (either mixed or pure), tissue (eg, endothelium, epithelium, mucosa or other tissue, including tissues containing the above CD4 + free cells), organ (eg, heart, lung) , liver, muscle, gallbladder, bladder, gonads, eye, and other organs), organ system (e.g., circulatory, respiratory, gastrointestinal, urinary, nervous, skin, or other organ systems) or organism (e.g. bird, mammal, etc.). It is preferred that the organs / tissues / cells to be targeted are part of the circulatory system (eg, including, but not limited to, the heart, bloodstream and blood, including white blood cells and red blood cells), the respiratory system (eg, nose, pharynx, larynx, trachea, bronchi, bronchi, lungs and the like), gastrointestinal tract (eg mouth, pharynx, esophagus, stomach, intestines, salivary gland, pancreas, liver, gall bladder and others), urinary system (eg kidney , ureter, bladder, urethra, and the like), the nervous system (eg, including but not limited to, the brain and spinal cord and specialized sensory organs such as the eye) and the skin system (eg, skin, skin and subcutaneous or skin cells) weaving). More preferably, the target cells are selected from the group consisting of heart, bloodstream, lung, liver, gall bladder, bladder and eye cells. The target cells need not be normal cells and can be diseased cells. Such diseased cells may be, but are not limited to, tumor cells, infected cells, genetically abnormal cells, or cells in the vicinity or in contact with abnormal tissue, such as tumor vascular endothelial cells.

Vector A "vector" is a nucleic acid molecule (typically DNA or RNA) that serves to transfer sequences of a transferred nucleic acid (ie, DNA or RNA) to a host cell. Three common types of vectors include plasmids, phages, and viruses. Preferably, the vector is a virus that contains encapsulated forms of vector nucleic acids and viral particles in which the vector nucleic acids are packaged.

Suitably, this vector is not a wild type strain I know and therefore contains man-made mutations or modifications. Thus, the vector is typically derived by genetic manipulation from a wild-type strain (eg, by deletion) to contain a conditionally replicating virus, as described below. Preferably, the viral vector comprises a virus strain that is of the same type as the wild-type virus causing the infection to be treated, which is preferably one of the aforementioned wild-type viruses. Accordingly, it is preferred that the vector be derived from an RNA virus, even more preferably it is derived from a retrovirus, and optimally, it is derived from a virus

human immunodeficiency. Such a vector derived from human immunodeficiency virus is generically referred to as a "crHIV" vector.

The vector is also preferably a "chimeric vector", i.e., linking the viral vector to other sequences, such as linking HIV sequences to one or more viruses (which are suitably derived from a wild-type strain of virus to contain a conditionally replicating vector). It is particularly preferred that the HIV sequences can be linked to sequences of a modified (i.e. wild type) adenovirus strain, adenovirus-associated virus, Alphaviridae virus, Flaviviridae virus, Hepadnaviridae virus, Papovaviridae virus, Parvoviridae virus, Herpesrviridae virus , a Poxviridae virus, a Paramyxoviridae virus, a Rhabdoviridae virus, a Paramyxoviridae virus, or a Retroviridae virus, including Onkoretroviruses, Spuma-retroviruses, and Lenti-retroviruses. Also included are viruses or genomes similar to viral genomes that are derived or associated with these virus families.

A chimeric vector is preferred when vector sequences (i.e., sequences encoding either proteins, fragments or non-coding sequences) derived from other than Lentivirus are inserted into the Lentivirus vector. It is preferred that they are inserted into an HIV-derived vector other than the HIV sequence.

As included herein, the vector may comprise either DNA or RNA. For example, either an RNA or a DNA vector can be used to derive the virus. Similarly, a cDNA copy can be made from viral RNA. Alternatively, cDNAs (or viral genomic DNA) portions may be transcribed in vitro to produce RNA. These methods are well known to those skilled in the art and are also described in the following examples.

A 'conditionally replicating virus' is a replication-defective virus that is defective only under certain conditions. Specifically, the virus can complete its replication cycle in a host cell that allows it, and cannot complete its replication cycle in a host cell that does not allow it. A "host cell" is a cell that is capable of being infected or is actually infected with a wild-type strain of virus or a pseudotypic vector. Such a wild-type virus infection may occur either before or after infection with a conditionally replicating virus of the invention. Alternatively, a "host cell" may be a cell that encodes wild-type virus or helper gene products that are essential for virus replication. Thus, the conditionally replicating vector of the invention is a virus (which is preferably of the same virus type as causing the infection to be treated) that replicates only when complemented with a wild-type virus (or helper) strain or when the wild-type virus infects cells which contain genomes of a conditionally replicating vector.

• · · · · · · • · · · · · · ·

In a preferred embodiment, the vector comprises an RNA virus (eg, a conditionally replicating HIV virus) that is introduced in the form of DNA. This preferred embodiment offers a method of using a replicating HIV-1 (crHIV) vector that provides non-pathogenic crHIV-1 vector genomes with a selective advantage over pathogenic wild-type HIV genomes. In particular, in cells containing both HIV and crHIV wild type genomes, crHIV RNAs have a selective advantage in enveloping into virions because they contain, for example, ribozymes that cleave wild-type RNA but not crHIV RNA. Such non-pathogenic crHIVs are capable of spreading to uninfected cells that are susceptible to HIV infection (eg, CD4 + cells) in the presence of wild-type helper virus. In this way, selective packaging and spreading of crHIV interferes with wild-type HIV replication.

Another preferred embodiment is a non-pathogenic crHIV vector which is non-pathogenic because it does not contain any combination of viral accessory protein sequences (such as, but not limited to, Vif, Vpu, Vpr or Nef or combinations or fragments thereof) that could make this vector pathogenic. These sequences may alternatively be present, but may be transcriptionally silent or non-translated. Optionally, the vector does not contain any combination of regulatory protein sequences (such as, but not limited to, Tat or Rev or combinations thereof) that would cause the pathogenicity of the vector. These sequences may alternatively be present but transcriptionally silent or untranslated.

However, the vectors preferably contain any combination of structural protein sequences (such as gag or fragment thereof), enzyme protein sequences (such as pol or fragment thereof) and / or envelope protein sequences (such as env or fragment thereof) in a form capable of being translationally active or quiet. Thus, the conditionally replicating vector may be able to replicate, but not at levels that are pathogenic to a given host organism. Instead, the vector needs complementation with a helper component (such as a helper vector) containing the necessary wild-type virus-derived sequences to replicate at levels sufficient to produce the desired therapeutic, prophylactic or biological effect. Determining the exact combination of proteins or nucleotide sequences present in the vector and the helper to obtain an optimal biological effect involves only the direct application of simple screening processes, including adding and removing different combinations of nucleotide sequences to the vector or helper construct, which are within the skill of the art. routine methods.

In addition, the aforementioned nucleotide sequences may be modified or mutagenized to alter their biological activity or, for example, reduce the possibility of their recombination with a helper construct. Numerous známy 9 · are known in the art.

methods for modifying or mutating vector and helper constructs to achieve more optimized constructs (eg, Current Protocols in Molecular Biology, Hartcourt Brace and Jovanovich, 2000; Molecular Cloning, Sambrook et al., Cold Spring Harbor Press, 1989 and Soong et al. Nature Genetics) 25: 436-439,2000, both of which are incorporated herein in their entirety).

The approach provided by the above vectors differs from the use of live-attenuated (LA) vaccines which use replication competent viruses lacking accessory proteins (see Daniel et al., Science, 258, 1938-1941 (1992)) and Desrosiers, AIDS Res. & Human Retrovir., 10, 331-322 (1994)), because LA vaccines do not make any effort to complement deficiencies that, for example, prevent effective elicitation of an effective immune response while maintaining safety. For example, it is known that multiple deleted LA SIV vaccines cannot elicit an effective immune response, while single deleted (Nef negative) LA SIV vaccines are pathogenic to juvenile cynomolgus monkeys (Baba et al,

1995).

An alternative approach to LA HIV vaccines provided by the above described invention would be to use at least two vectors, at least one of which is a multiple deleted HIV vector and the other (helper) vector expresses all accessory proteins except Nef. Thus, the multiple attenuated HIV vector could replicate in a conditional manner without causing disease, being complemented in a controlled manner with Vif, Vpr and Vpu from the helper vector. The first vector may be engineered to contain genetic antiviral means to interfere with the replication and spread of wild-type HIV. Alternatively, this first vector could be used to elicit an effective immune response against a wild-type virus. It will be clear to those skilled in the art that a simple screening process will allow evaluation of which true combination of sequences deleted from the first vector but present in the helper vector would allow the first vector to optimally deliver a therapeutic or prophylactic response while maintaining safety by its non-pathogenicity.

Another preferred embodiment of a conditionally replicating vector-helper system may be the use of an HIV-1 vector for expression of gag, pol, tat and rev, using an HIV-2 derived helper vector, for example, for expression of the Vif, Vpu, Vpr genes and gene. The invention is not limited to this example since any combination of the above-mentioned genes located in any combination of compatible vectors, including reverse HIV-1 and HIV-2 or chimeric HIV formats, is equally possible. Expression of Tat from the HIV-1 background could transactivate both HIV-1 and HIV-2 LTRs to complement each other to form ····

0004

0

Vector portions that contain either the HIV-1 vector genomes or the HIV-2 vector genomes. However, these two genomic RNAs cannot effectively dimerize, thereby preventing the simultaneous localization of both vector and helper genomes that could otherwise be packaged together in a single virion particle. In the case of co-packaging into one virion particle, during reverse transcription, after that particle infects another target cell, recombination between the genomes of the vector and the helper could occur that would produce a replication competent vector (RCV).

As a further case of possible production of co-packaged constructs, these vectors may further comprise one or more nucleic acid sequences that reduce the risk of any recombination. For example, the first vector may comprise a ribozyme (or antisense / ribozyme) sequence specific for cleavage of the helper vector. Co-location of these vectors at the same cellular, cellular or extracellular site could result in cleavage of the helper vector to enhance safety in diminishing or co-location of these vectors, resulting in inactivation of the genome after recombination or otherwise preventing RCV production. This approach can be altered by including a ribozyme on the helper vector, or further improved by including ribozymes on both vectors to further enhance safety.

Alternatively, antisense molecules can be used to replace the above ribozymes, so that double-stranded hybrid RNA structures are rapidly degraded by cellular endonucleases. Another preferred embodiment is that the antisense sequences may be greater than 16 bases in length of the ribosomal RNA used for targeting.

In a modified approach, the helper virus may be derived from a heterologous virus (for example, any of the viruses and virus classes listed above but preferably derived from adenovirus, adenovirus-associated virus, mouse oncoretrovirus or non-HIV lentivirus), which may be proteins expressed constitutively. This heterologous helper vector may alternatively be designed to utilize the HIV-LTR for inducible or self-regulated Tat expression to transactivate the expression of the HIV-1 vector. The advantage provided by the heterologous viral helper is the restrictive association between the genomes of the first and second vector, thus further reducing the possibility of recombination and the formation of a possible RCV.

In another embodiment, the vector comprises an antisense sequence that is present in the helper genome or inserted into the helper genome. A non-limiting example is seen in the vector pN1cptASgag, which is analogous to the vector pN1cptASenv, except that the antisense sequence is directed to the gag sequence present on the helper vector in addition to or instead of the env sequence present in wild-type HIV. This anti-gag antisense sequence is placed in front of the acceptor 9 9 9

9 9,999 9 9 9

9 9 9 9 9

9 9 9 9 9

99 99 999 99 99 splice site present after the RRE sequence present in the vector. Therefore, the gag sequence will only be packaged in genomic and not subgenomic or spliced vector RNAs. Thus, intron-containing helper genomes, such as VIRPAC helpers, will preferentially target the cell splicing complex, while RNA genomic vectors that contain anti-gag antisense sequences will preferentially bypass this splicing mechanism.

Differential transfer of vector and helper genomes within a cell should have minimal effect on vector titer, even if vector and helper vectors are randomly placed together, since base-pair hybridization of vector and helper genomes should result in inactivation or destruction of such vector- and vector-containing particles. helper genome and prevent recombination of the vector with the helper, resulting in RCV. In an alternative embodiment, the helper may be constructed to contain antisense sequences of an anti-U5 vector directed to U5 sequences that have been found in the conditionally replicating vector. Without limiting the spirit of this aspect of the invention, anti-U5 antisense sequences may be inserted distal to the helper coding sequences but before the transcription termination site. Therefore, if vector and helper RNAs are randomly co-located, packaged, and optionally recombined, these antisense sequences will then destroy or inactivate such co-packaged viral particles and prevent recombination.

In yet another embodiment, the helper may comprise target sequences for the first nucleotide sequence present on the vector. A non-limiting example occurs when the env fragment of the correct orientation is placed in the helper of a bp plasmid pair that contains the vector pN1cptASenv. Thus, if both the vector and the helper RNA are co-located, then a correctly oriented env sequence in the helper should undergo base pairing with the antisense env sequence in the vector, thereby preventing recombination. The above examples are not intended to limit the present invention to this one or two types of genetic antiviral sequences. It will be appreciated by those skilled in the art that numerous genetic antiviral sequences, as well as their related target sequences, may be inserted into vector and / or helper constructs. Another non-limiting example is that the pNlcptASgagASenv vector and helper, which contain the correctly oriented gag and env fragment sequences, can be used in combination to increase safety by reducing the likelihood of co-packaging and recombination to form RCV.

In another preferred embodiment, for expressing vector and helper components, the components are transiently expressed. One way of ensuring that the vector and the helper transiently express their genome is by constructing the vector or this helper.

4000

0 · 0000

Use of, for example, an integrase of the negative Pol gene. Such lentiviral integrase mutants are known in the art and have been reported not to be infectious (Hirsch et al 1989 Nature 341: 573-574). The vector and / or helper genomes that are thus produced by integrase negative production cells will not integrate, but can be expressed in a transient manner and thus regulate the extent of replication of the vector or helper. Yet another way to transiently express either a vector or a helper is to disrupt the AAT sites in the vector LTRs. These sites are responsible for the active integration of the reverse transcribed vector genome DNA into the host cell chromosome.

Another embodiment of the above uses a fusion protein to coat a functional integrase molecule into a viral particle. In this embodiment, neither the conditionally replicating vector such as the lentivector nor the helper vector will encode the integrase, but the functional integrase fusion protein will be available due to expression from another plasmid construct such as the helper vector or from an integrated copy in the packaging cell used, and provides functional integrase activity after infection. Alternatively, this integrase protein is obtained in trans expression from a helper construct that encodes it. In this embodiment, the conditionally replicating vector or lentivector cannot encode an integrase. Said fusion protein may be, but is not limited to, a vpr-integrase protein that comprises a protease cleavage site at the junction between the vpr and the integrase amino acid sequence.

The foregoing description of the double-vector helper system is by no means a limitation of the present invention to dual vector constructors or helpers. Any combination of two, three, or more vectors and / or helper can be used to obtain the components necessary to produce the vector. The division of the necessary genomic elements into multiple vector or helper components has an effect on increasing safety, since the formation of RCV recombinations is more difficult for multiple vector and helper genomes. Safety can be further enhanced by constructing the vector (s) or helper (s) to contain little or no regions of homology between them, which is another preferred embodiment. Splitting the necessary genomic elements into multiple components can also further limit the replication of the conditionally replicating vector, since it is unlikely for more than two genomes, compared to only two, to be present in the host cell at the same time. The optimal number of vector (s) and helper (s) needed can be readily determined by simply screening the various combinations and can vary depending on the particular viral vector system being used.

One simple, but non-limiting, example of limiting or removing regions of homology between the vector (s) and the helper (s) is the simple degenerate nucleotide to toto this ** ♦ * »· ♦» ··> *

9 9 9 9 9 99 · · 9 · ···

9 9 9 9 9

9 9 9 9 9

999 99 99 9 99 9 9 99 sequence, maintaining the encoded amino acid sequence. Methods of degenerate sequences are well known in the art and are routine. One preferred method is to humanize these sequences when therapeutically used in humans. The codon used has been tabulated for primates and is described in Wada et al. (Nucleic Acid Research vol. 28 Supplement: 2367-2411, 1990), which is hereby incorporated in its entirety).

Degeneration can also be used to protect the helper construct from the effects of any agent designed to target the vector to be packaged with the helper. This can simply be done by degenerating the same target sequence, if any, as found on the helper construct. In addition, degeneration of both vector and helper constructs can be determined to reduce recombination with other viral sequences, including sequences of other wild-type, which can be found simultaneously with the vector or helper sequence in the cell. For example, the use of a vector-helper pair in an HIV-1 and HIV-2 based coating system can be modified such that the vector-helper pair is also degenerate with respect to the outer retro-element. Degeneration of the nucleotide sequence of either the vector or the helper will reduce the co-location of the putative recombinations and thus reduce the risk of developing a replication competent virus between the vector and helper genomes or between the vector or helper genomes and a competing genome such as an endogenous retro-element.

In particular, crHIV genomes are introduced into virus-infected cells or uninfected cells. Infected cells supply the crHIV genome with proteins required for encapsulation and production of viral virions. The crHIV genomes are introduced into uninfected cells preferably either directly by transduction (e.g., this can be done, for example, by crDNA transduction by liposomes or by using a chimeric viral vector) or by infection with crHIV particles that result in transfection of cells infected with wild type HIV. Uninfected cells containing the improved crHIV vector of the invention do not produce a number of crHIV portions that are pathogenic to the host. Some embodiments of crHIV vectors of cells that are not superinfected with wt-HIV will produce a certain amount of crHIV portion but at levels that are not pathogenic to the host. Cells containing a vector of the invention may remain susceptible to superinfection with a wild-type virus that delivers the proteins needed to further produce crHIV particles. In this sense, also, the conditionally replicating vector of the invention, in the presence of complementary wild-type superinfection, functions as a type of "viral delivery vector" that can, for example, be followed by further cycles of crHIV infection (i.e. in the presence of competing wild-type HIV). Such a vector is a source of virus for more than one cycle of viral replication, and thus infection of other cells or many cycles. * * • · · · · · · - ** · 9 9 9 φ 5 · φ

III replication at levels that can elicit a biological response, such as an immune response. This improvement is in contrast to other vectors, such as those used in standard enveloping cell lines, that serve only one or multiple replication cycles that are either insufficient to elicit a corresponding biological response or are pathogenic to the host.

If desired (e.g., to facilitate the use of this vector in vitro), wild-type virus gene products can be delivered to the infected cell along with the conditionally replicating vector. Wild-type virus gene products can be delivered not only by co-infection with the wild-type virus strain (or cDNA or for RNA virus virus), but also by delivery to the cell in the form of their genes subcloned in an expression vector ("helper" or "helper vector") ) which is capable of participating in the transcription or translation of host cell sequences (regulatory or structural) or, alternatively, gene products may be delivered externally, i.e., by adding protein products to the cell.

For example, a crHIV vector containing all wild-type HIV proteins, except, for example, the Tat coding sequence, can be constructed. Instead of using a helper expression construct that expresses Tat, the Tat protein itself can be used as a helper. The advantage of using this protein in place of the helper construct is that there is no theoretical possibility of wild-type virus formation because no nucleic acid sequences are available for recombination. Thus, crHIV can be propagated using Tat protein and not by helper nucleic acid constructs per se. Tat 1 or Tat 2 (corresponding to the first or optionally the first and second exons of Tat) can be used as helper. Methods for producing and purifying such proteins are well known in the art.

In one preferred embodiment, a chimeric Tat protein is used. Tat should be incorporated into the fusion protein (eg Tat-VP16) and shown to maintain its transactivating activity of the HIV-LTR promoter. Other chimeric Tat proteins may be constructed to enhance the desired biological effect. For example, if enhancing the immune response is a desired biological effect, a Tat-GM-CSF fusion protein can be constructed. This approach is not limited to one type of chimeric protein and it may be desirable to add at least a second chimeric (or non-chimeric) Tat protein (e.g., Tat-IFN-α, Tat-TNF-α, Tat-IL-4, Tat-G-CSF, TNF-α, GMCSF, IL-4, G-CSF, IFN-α (to give just a few options). It will be apparent to one of ordinary skill in the art to construct and screen other chimeric proteins and screen them for the ability to enhance the immune response while maintaining the helper function for the vector. Another preferred embodiment is, for example, the Tat-Rev fusion protein construct or the native HIV Tat-Rev fusion protein, Trev. The above description of chimemic Tat proteins

therefore, it is not limited to the inclusion of cytokines or cellular proteins only, but viral proteins (producing homologous or heterologous viral fusion proteins) may also be used according to the invention, depending on the desired biological effect.

With respect to a "helper vector", its expression may be cell-specific or cell-nonspecific and introduced into a host cell in accordance with a conditionally replicating vector as defined herein, and thus, allow continuous replication of the conditionally replicating viral vector.

The "helper vectors" of the invention can deliver the necessary gene products for replication by either a single vector or multiple vectors. Such vectors are preferably used in a transient transfection system. Alternatively, these gene products may also be incorporated into the genome of a host cell or cell line. In accordance with the present invention, a simple vector or plasmid (which may be mono, bi or multicistronic in the host cell) is preferred due to the increased titers due to the increased likelihood of co-transfecting the helper vector and the conditionally replicating vector into the same cell. The reduced likelihood of co-transfecting more than two different vectors into the same cell makes the use of a simple helper vector even more desirable. Reducing the number of vectors or plasmids involved in the transfection system is also more cost effective, since it is not necessary to produce as many plasmids to coat retroviral constructs as compared to a conventional three-plasmid transfection system.

"Complementation" as used herein refers to the non-genetic interaction of viral gene products from various sources in a cell. Specifically, in a mixed infection, complementation involves an increase in virus yield of one or both of the parental genomes, while the genotypes of the parental genomes remain unchanged. Complementation can be non-allelic (ie, intergenic, where mutants defective in different functions assist each other in viral replication by delivering a function for which another virus is defective) or allelic (ie, intragenic, where two parents have defects in different domains of the multimeric protein).

Desirably, the types of cells that can be transfected (transduced) with crHIV DNA (i.e., using liposomes or using a heterologous vector to prepare a chimeric vector as described above) may be cells either infected or uninfected with HIV. Cells infected with HIV may be activated or not activated. When activated, they can immediately transcribe wild-type HIV RNA and crRNA, resulting in selective packaging of crHIV RNA into the progeny virions. If HIV-infected cells are not activated, crHIV DNA will remain in them unless activated (eg by stimulation with mitogens, antigens, and the like), resulting in selective coating again • ···· ·· ···· ·· · · 9 9 999 99 9

9 9 9 9 9 9 9 9 9 ··· ···················· CrHIV RNA into virion progeny. Activated and unactivated uninfected cells that are transfected with crHIV DNA will not produce virions unless they are superinfected with wild type HIV and activated by stimulation, again resulting in the selective packaging of crHIV RNA into the progeny virions.

One embodiment of a host cell transduced with a vector of the invention is when the cell contains a maximum copy number of the vector that is not significantly toxic to either the host cell or the host organism containing the cell. Another embodiment of a host cell transduced with a vector of the invention is when the cell contains at least one copy of the vector, but preferably contains the minimum number of copies of the vector required to produce a biological effect. This number of copies in the cell can be determined, for example, by TaqMan PCR and an acceptable copy number or copy range suitable for both the desired biological effects and lacking toxicity can be readily determined by one skilled in the art. Acceptable copy numbers will vary depending on factors including the type of cell to be transduced, the nature of the vector (eg, viral origin) and the desired biological effect, but can be readily determined by routine screening by one skilled in the art.

Superinfection of cells containing crHIV genomes (eg as a result of transfection or infection) occurs because crHIV genomes do not encode viral proteins that block superinfection (such as env and nef). The resulting crHIV virions can infect uninfected cells because these viral particles contain reverse transcriptase molecules that carry all HIV particles so that they can generate DNA provirus from their genomic RNA. This process is called reverse transcription. If crHIV virions infect uninfected cells, they may undergo reverse transcription and produce a provirus from their genomic RNA. Thus, these cells are equivalent to uninfected cells that are directly transduced with crHIV DNA. They cannot produce crHIV particles until they are superinfected with wild-type HIV and are activated, and then selective packaging of crHIV RNA into the progeny virions occurs again. It is possible that crHIV particles may also infect some cells that are already infected with HIV (see, eg, Yunoki et al., Arch. Virol., 116, 143-158 (1991); Winslow et al., 196, 849-854 (1993)). Chen et al., Nuc. Acids res., 20,4581-4589 (1992) and Kim et al., AIDS Res & Hum. Retrovir., 9, 875-882 (1193)). For this to happen, however, these HIV-infected cells must not express proteins that reduce CD4 expression, as this would prevent crHIV virions from infecting the cells. Activated, HIV-infected cells generally regulate down-regulation of CD4 expression. Accordingly, HIV-infected cells that are not activated are potentially susceptible to crHIV superinfection and may thus be another source of crHIV particle production.

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

A preferred crHIV vector of the invention comprises the sequences required for RNA transcription, tRNA primer binding, dimerization and packaging, and lacks either sequences encoding proteins that block superinfection with wild-type HIV (eg, nef or env proteins), but these sequences the sequences are either not transcribed or not translated into a functional protein, so their expression is considered "silent". Even more preferred is a vector that lacks a region or sequence that encodes a wild-type HIV region, starting with the gag coding sequence up to and including the nef gene. However, it is optimal if the vector contains a rev responsive element (RRE) that is cloned into the vector in the deletion region or some other conventional region. Such a preferred HIV vector is termed "lacking a region or sequence encoding that region" when it can be administered in its RNA form or alternatively in the DNA form as described above.

In accordance with the present invention, in order to achieve successful complementation in a system that utilizes a helper vector in conjunction with a conditionally replicating vector, the helper vector must provide any viral component necessary to envelop the virus but which is not expressed by the conditionally replicating vector. Thus, if two or more different vectors are completely complementary to each other with respect to the necessary viral components, then the composition of the individual vectors is subject to many permutations. Each of these permutations is intended by the present invention. By way of example, the gag and pol genes necessary for successful packaging and replication can both be incorporated into either a helper vector or a conditionally replicating vector, or both can be in either or both of these vectors. In addition, a single helper vector construct may comprise both gag / pol and env sequences, including heterologous env sequences, under the control of a single set of transcriptional regulatory elements, including the promoter, or under the control of separate promoter elements. For example, LTRs may be used to express gene products in a vector of the invention. The use of various promoters, including inducible promoters, guarantees, for example, the possibility of varying the regulation of gag / pol and env sequences. Thus, an env sequence, particularly a heterologous env sequence, such as a VSV-G sequence, can be expressed at a higher level.

The construction of the vector is well known to those skilled in the art. These methods can be used to construct both the conditionally replicating vector and the helper vector. As used herein, the term "vector" refers to both types of vector. Furthermore, the term vector includes any lenti viral vector without necessarily being a conditionally replicating vector. Such vectors may also be referred to as generic lentiviral vectors. In a preferred embodiment of the invention, however, the vector is a conditionally replicating lentiviral vector. For example, as described in Example 1, the DNA form of an RNA virus, such as

HIV, cleaved by restriction enzymes to excise the HIV coding sequence from the gag coding region to and including the U3 region, the following nef gene. A cloning envelope containing a polylinker that contains multiple restriction sites is inserted into the deletion region before being ligated to form the usual restriction sites for cloning in this vector. The DNA fragment containing RRE is subcloned into one of these sites. The resulting vector produces a disrupted gag transcript and does not produce wild-type Gag protein or any other wild-type HIV protein. In addition, it is not necessary that this vector express even a truncated gag protein if the gag translation initiation sequence can be mutated to prevent its transcription.

Using the same approach, crHIV sequences can be linked to other sequences, from the same or a different virus or vector, to derive a chimeric vector as described above. For example, crHIV sequences can be ligated to sequences from randomly named Sindbis virus, AAV, adenovirus or amphotropic retroviruses. An additional viral sequence that can be used includes sequences from Herpes, Pox, and other viruses described herein, including a virus that can be used to ensure transmission of crHIV sequences. Such a chimeric vector may be introduced into the cell either by using a coupled viral mechanism for cell entry (eg, adenovirus receptor-mediated endocytosis) or by other means, eg, liposomes.

Preferably, according to the invention, the vector (i.e., the conditionally replicating vector, which is preferably the crHIV vector) comprises at least one nucleic acid sequence whose inclusion (ie, presence, transcription or translation) represents a selective advantage. There are two types of such nucleic acid sequences that are intended to be included in this vector: (1) a nucleic acid sequence whose inclusion optimally constitutes a selective advantage for viral replication and extension of a vector containing such a sequence relative to the wild-type virus strain (i.e. and (2) a nucleic acid sequence, the inclusion of which optimally constitutes a selective advantage for a cell which is infected with a vector comprising the sequence as compared to the cells infected with the vector. or an uninfected wild-type strain I know (i.e. preferably the wild-type strain from which the vector was derived (as well as, for example, a helper expression vector that promotes vector replication and / or function in an uninfected host cell) and cell survival, graduation producing and / or propagating the vector particle by promoting production of crHIV vector virions from crHIV vector producing cells by inducing apoptosis, facilitating the production or promotion of immunological function or targeting to achieve the desired prophylactic, therapeutic or biological effect of the result. Each or many of these sequences, i.e. sequences which, alone or in combination with other factor (s), promote vector propagation and / or promote the appropriate function of the host cell so as to permit a favorable prophylactic, therapeutic or biological outcome, it may be contained in a vector, either in the absence or in the presence of other sequences, i.e. the vector may comprise "at least one nucleic acid sequence" and "at least one additional nucleic acid sequence".

A third type of nucleic acid sequence is a sequence that represents a host cell phenotype that shrinks, minimizes, or prevents viral infection. Such nucleic acid sequences include sequences that encode a gene product that, when expressed, protects the host cell from viral infection. For example, individuals homozygous for the Δ32 allele of the CCR5 protein were protected against HIV-1 infection due to the absence of a major co-receptor for HIV-1 entry into cells, by expression of the disrupted CCR5 protein on their surface. Studies with heterozygous individuals have shown that the Δ32 allele has a transdominant effect on cell surface expression of wild-type (dt) CCR5. This may be due to the ability of the Δ32 allele to dimension with the dt protein and prevent proper cell surface expression.

The Δ32 allele contains a deletion of 32 nucleotides, causing a mutation of the lattice leading to a disrupted CCR5 protein. Since this violation concerns 31 amino acids at the C-terminus of the Δ32 allele that are not present in the dt protein and can produce an undesired immune response when present on the cell surface, the present invention encompasses the expression of a disrupted mutant CCR5A32 protein (CCR5-32T). potentially antigenic amino acids. This protein retains its ability to impart a transdominant effect to obtain cells protected against HIV infection. This CCR5-32T coding sequence is readily generated by introducing a stop codon immediately after the last amino acid common to both dt CCR5 and CCR5A32 (after tyrosine at position 184).

In another embodiment, another third type of nucleic acid sequence is one that further enhances the transmitted phenotype of shrinking, minimizing, or protecting against viral infection. Such a nucleic acid sequence may encode a genetic antiviral agent that, for example, targets a cellular gene. For example, the vector may express the aforementioned CCR5A32T mutant protein and additionally express an antisense or ribozyme molecule, from the U1 promoter and contained in the U1 snRNA (described in US Patent 5,814,500), which is, for example, targeted to the wild type CCR5 molecule. The antisense region that targets wild type CCR5 will degenerate in the vector-borne CCR5A32T sequence and produce a transdominant mutant RNA resistant to the effects of anti-CCR5 antisense or ribozyme molecules, but will be translated into the correct amino acid sequences to produce a functional CCR5A32T protein.

The above method of co-expressing a ribozyme or antisense region targeted to a gene product and degenerating the gene at target sites to prevent binding or cleavage of the ribozyme or antisense region is not limited to CCR5 or a molecule that can minimize viral replication. Another non-limiting example of therapeutic application is the targeting of an oncogenic protein, such as the Bcr-Abl fusion protein, which is an etiological agent for chronic myelogenous leukemia. A vector construct containing the Ber or Abl gene or both may express these genes simultaneously with the expression of an antisense region or ribozyme that is targeted to any site along the Bcr-Abl transcript. This abnormal Bcr-Abl fusion transcript will then be destroyed while the native Ber or Abl or both will be expressed to remove this abnormal defect. Thus, the Ber or Abl genes have a selective advantage in enhancing wild-type or abnormal Bcr-Abl fusion gene. A preferred vector will express the Ber and / or Abl construct from their native promoters, while anti-Bcr-Abl ribozyme or antisense sequences will be expressed from the U1 promoter and included in the U1 snRNA sequences.

The above example of Bcr-Abl does not limit this invention to oncogen targeting, and it will be apparent to one of skill in the art that this approach could be used for the therapeutic or prophylactic substitution of any abnormal gene, unwanted gene or even desirable gene (s) of interest. The target gene may be homologous or heterologous to a modified gene that is resistant to the effects of this genetic antiviral molecule. Other disease states that may be the target of this method are apparent to those skilled in the medical and clinical art. Molecular targets for the treatment of blood diseases according to the method described herein are described, for example, in The molecular basis of blood diseases (Stamatoyannopoulos, Majerus, Perlmutter & Varmus, 3rd ed., Philadelphia: WB Saundres Co., copyright 1987, 1994 and 2001) , which is hereby incorporated by reference).

The present invention should not be limited to clinical applications. This approach can be used to determine the function of the gene sequence of interest. For example, a gene sequence of interest modified in a region of interest may be co-expressed with an antisense or ribozyme sequence that inhibits or disrupts the sequence of all unmodified gene by targeting the corresponding unmodified region. Thus, the sequence of interest of the modified gene will have a selective advantage over enhanced expression against the sequence of the modified gene. The unmodified gene allows the determination of the function of the sequence of the modified gene or the gene product encoded therein by analytical methods known in the art.

The term "nucleic acid" has the previously described meaning. Specifically, a "nucleic acid sequence" includes any gene or coding sequence (i.e., DNA or RNA) of potentially any size (i.e., limited by any enveloping constraints imposed by the vector), the presence of which provides a selective advantage as further defined herein. A "gene" is any nucleic acid sequence encoding a protein or nascent mRNA molecule (regardless of whether the sequence is transcribed and / or translated). While the gene includes coding sequences as well as non-coding sequences (eg, regulatory sequences), the "coding sequence" contains no non-coding DNA.

A nucleic acid sequence, its presence carries a selective advantage to a host cell for a vector carrying this sequence, relative to a wild-type virus strain or competing genomic sequence that would interfere with or affect the amplification of the vector.

The nucleic acid sequence that confers a selective advantage of the vector in the host cell over the wild-type virus strain is preferably any sequence that allows viral particles that are propagated by the vector to be selectively produced or enveloped over viral particles that are reproduced by wild-type virus. These sequences include, but are not limited to, a sequence that results in an increase in the number of vector genomes produced intracellularly compared to the wild-type genome, and an antiviral nucleic acid sequence. These sequences also include, but are not limited to, a sequence that results in an increase in the number, property, or status of a vector genome that is produced intracellularly compared to wild-type genomes that are not derived from a related wild-type virus.

The first category of nucleic acid sequences that confer a selective advantage in a host cell on a vector containing this sequence as compared to a wild-type virus strain are sequences such as a promoter. A "promoter" is a sequence that directs RNA polymerase binding and thereby promotes RNA synthesis, and which may contain one or more enhancers. "Amplifiers" are cis-acting elements that stimulate or inhibit the transcription of adjacent genes. An enhancer that inhibits transcription is also referred to as a "silencer". The 'silencer' can also be an 'isolation' element, as is the case with the DNA-hypersensitive sites of the chicken erythroid isolation element. Enhancers differ from DNA-binding sites for sequence-specific DNA binding proteins found only in the promoter (also referred to as

9 9 9 9 • 9 9999

··· “promoter elements”), in that the amplifiers can operate in both directions and at distances of up to several kilobases (kb), even from a downstream position.

Accordingly, and preferably, the promoter (e.g., the long terminal repeat region (LTR)) of the conditionally replicating HIV vector) is modified to render the vector more sensitive to certain cytokines than the wild-type strain of HIV. For example, a modified HIV promoter is available that demonstrates increased transcriptional activity in the presence of interleukin-2. The incorporation of this promoter into the vector and the introduction of this vector into the HIV-infected wild-type cells preferably results in increased production and packaging of progeny virions of the vector genome as compared to the wild-type HIV genome. Other cytokines and / or chemokines (eg, including, but not limited to, interferon-α, tumor necrosis α-factor, RANTES, and the like) can likewise be used to promote selective packaging of virions encoded by the vector. The placement of elements that render the cytokine-responsive HIV-LTR in no way limits the scope of the invention to these embodiments. Any nucleotide sequence can be inserted into the HIV-LTR to modify the sensitivity of this promoter. A list of factors that bind to DNA sites that can be inserted into the HIV-LTR can be found at http://transfac.gbf.de/TRANSFAC/lists/browse.html, available from September 8, 2000, which is hereby incorporated by citation in its entirety. The list of DNA-binding factors for Homo sapiens (http://transfac.gbf.de/TRANSFAC/lists/factor/species/humanHomosapiens.html), accessible from September 8, 2000, is likewise incorporated by reference in its entirety. .

Similarly, silencers or isolators may also be inserted into promoter or enhancer regions of a native or modified retroviral LTR, such as HIV-LTR, to precisely regulate expression by this promoter. In certain cases, elements introduced into HIVLTR may present a selective disadvantage to this vector with respect to infection by wild-type virus. However, the purpose of applying this type of vector may be to express the nucleic acid sequence of interest ("useful gene"), which, for example, inhibits HIV replication without competition, instead of competing with the wild-type virus in enveloping the virions of the progeny. In this case, the first nucleic acid sequence does not confer a selective advantage on the vector, but must at least, for example, inhibit recombination between the vector and the helper during the vector production process.

A second category of nucleic acid sequence that confers the selective advantage of a vector comprising this sequence over a wild-type virus strain comprises, as a preferred nucleic acid sequence, an antiviral nucleic acid sequence. "Antiviral agents" are categorized according to their mode of action, eg as reverse transcriptase inhibitors, competitors of virus entry into the cell, vaccines, protease inhibitors, and genetic antiviral agents. "Genetic antiviral agents" are DNA or RNA molecules that are transferred into cells and affect their intracellular targets either directly (ie, as they were introduced intracellularly) or after their conversion to either RNA or protein (see review by Dropulič et al. ( 1994), supra). Genetic antiviral sequences are also preferred nucleic acid sequences. Genetic antiviral agents include, but are not limited to, antisense molecules, decoy RNAs, transdominant mutants, toxins, RNA modifiers and modulators, and protein splicing, EGS (alone or in direct association with M1, U1, PRE or CTE elements), immunogens, RNAi (see Overseas et al. Cell 101: 25-33, (2000)), nucleotide sequences or molecules that modify or modulate splicing, constitutive transport elements (CTE), nuclear export and import factors, DNA integration factors, RNA stabilizing or destabilizing elements, post-transcriptional regulatory elements (PRE), proteins that interfere with virus replication, interferons, toxins, immunogens (defined by any gene that is immunologically related), antibodies (full-length antibodies, single-chain antibodies or ligand-displaying molecules), ribozymes or any element that affects any aspect of vector or wild-type virus replication, and ribozymes. Desirably, the genetic antiviral agent is an antisense molecule, a transdominant mutant, an immunogen, and a ribozyme. Thus, a preferred nucleic acid sequence that confers a selective advantage over a wild-type virus strain on the vector is an antiviral genetic agent sequence selected from the group consisting of an antisense molecule, an immunogen, and a ribozyme.

Genetic antiviral agents, as used in the present invention, are limited to targeting viral sequences, particularly when they are not located in conditionally replicating viral vectors. As a non-limiting example, the genetic antiviral sequence may be placed in a lentiviral vector such that the agent is directed to viral or non-viral targets, for example, cellular, bacterial, or parasitic targets. In this case, the lentiviral vector comprises a gene of interest operably linked to an unmodified or modified HIV-LTR (to the expressed spliced or uncut RNA) and, in addition, a genetic antiviral agent or sequence that is linked to a promoter sequence that is not operably linked to HIV-LTR. .

A preferred sequence is antisense sequences or ribozymes that are chimeric to snRNAs, preferably U1, U2, U3, U4, U5 or U6 snRNAs.

The genetic antiviral agent is also not limited to a single sequence or a single type of sequence present in either the vector or the helper. Two or more agents, either located distally or preferably joined in tandem, may be present in the vector or helper construct simultaneously. It is preferred that two or more different antivirus '

* 9 9 9 9 9 9 9 999 9 9 999 9 9 9 999 999 9 genetic agents. Another embodiment of the genetic antiviral agent for use in the invention is any one that binds two different types of genetic antiviral agents.

An "antisense molecule" is a molecule that, based on the rules of base pairing and availability of volatile base pairing, is a "mirror image" of the short segment of the gene whose expression is to be blocked. The antisense molecule directed against HIV hybridizes to wild-type HIV RNA, thus allowing its preferential degradation by cellular nucleases. The antisense molecules are preferably DNA oligonucleotides, suitably from 20 to 200 base pairs in length, preferably from 20 to 50 base pairs in length and optimally less than 25 base pairs in length. The antisense molecule can be expressed from crHIV RNA, which preferably binds to wild-type genomic RNA, thereby providing crHIV RNA with a selective advantage for enveloping into progeny virions.

Antisense molecules expressed in vectors as RNA are preferably at least 20 bases to any length, but preferably up to 2000 base pairs in length, more preferably 50 to 500 base pairs in length. These antisense molecules preferably bind to wild-type genomic RNA and not to vector RNA, thereby providing a vector with a selective advantage over wild-type virus. Preferred regions for the formation of an antisense molecule in an HIV virus-derived vector are, for example, the envelope (env) region, the Tat or Rev protein region, the accessory gene region (Vif, Nef, Vpr, Vpu), the Gag region 3 'relative to a Nsi I restriction enzyme site on pNL4-3, and a Pol region that does not contain a Pol 545 base region as shown below.

"Immunogen" refers to any immunologically related gene and gene product encoded by it. Although historically the term "immunogen" has been used in reference to an adjuvant used in vaccination, the terms "immuno-" and "gene" are used herein to refer to an immunologically related gene and its gene product encoded therein. For example, the immunogen may encode a single chain antibody (scAb) directed against a viral structural protein, or it may encode an antibody that is secreted by the cell extracellularly. The immunogen is transferred as a nucleic acid and is expressed intracellularly. Similarly, the immunogen may also encode any antigen, surface protein (including those that are class restricted), or a signal antibody that facilitates selection of the vector and / or the host cell. In a preferred vector, the nucleic acid sequence comprises a scAb coding sequence that binds to the wild-type HIV Rev protein. This advantageously prevents the maturation of the Rev protein by leading to its retention in the endoplasmic reticulum. Specifically, Rev proteins, alone or in conjunction with other cellular factors, export uncut and simply spliced HIV RNA from the nucleus into the cytoplasm by binding to RRE (a highly structured cis-acting RNA target sequence for Rev, called a Rev-sensitive element) and then oligomerize and surround and HIV RNA. HIV RNAs that are complexed with Rev are exported to the cytoplasm and bypass the mechanism

4 ’# 0

0 »00 * 0 ·· 0003 • 0 000« * 0 0 * 0 0 000 0 ··

0000 »000 0 000 00 0 0> 0>

0 4Λ 0 0 00 00 00 00 cell splicing. Thus, if wild-type Rev does not bind to wild-type RRE, then wild-type HIV RNAs are not exported to the cytoplasm and are not encapsidated into progeny virions.

Preferably, the vector containing the scAB nucleic acid sequence further comprises a modified RRE sequence and encodes a mutated Rev protein that recognizes a modified but not wild-type RRE. Accordingly, in cells that contain wild-type HIV and a vector that contains a scAb nucleic acid sequence, the vector is preferably packaged into virions. A similar strategy is advantageously used when the target for wild-type scAB proteins is an HIV matrix or nucleocapsid (or any protein involved in protein / RNA interactions that affect encapsidation of viral RNA).

A "ribozyme" is an antisense molecule with catalytic activity, ie, an RNA binding site and inhibiting translation, binds RNA ribozymes and affects site-specific cleavage of bound RNA molecules. Generally, there are four groups of ribozymes: the intervening sequence of Tetrahymena Group I, EGS (external leader sequence), and hammerhead and hairpin ribozymes. However, there are also other catalytic motifs in other RNA molecules (e.g., hepatitis delta virus and ribosomal RNA in fungal mitochondria).

A preferred ribozyme is a ribozyme in which the catalytic domain cleaves a 3'nucleotide sequence of NUH, where N can be any nucleotide (ie, G, A, U or C) and H can be either A, C or U. is most efficiently cleaved by this ribozyme, instead of the GUC, then the NUH sequence preferably comprises a GUC site.

Desirably, such a ribozyme cleaves in the wild-type region of the virus strain or its transcripts, but does not cleave in the region of the vector or its transcripts. The ribozyme cleaves the virus or its transcripts in the sense that the virus or vector may be either RNA or DNA, as previously mentioned. By " in-region " is meant cleavage in the target region, i.e. preferably in the region of the virus that is necessary for virus propagation. Desirably, the vector is modified such that this particular target region (if present in the vector at all) is not cleaved by the ribozyme. Optionally, the ribozyme may cleave the vector if the cleavage does not take place in a region that is required for multiplication of viral, e.g., crHIV particles.

Preferably, the ribozyme is encoded by a sequence selected from the group consisting of SEQ ID NO: 3 (i.e.

CACACAACACTGATGAGGCCGAAAGGCCGAAACGGGCACA) and SEQ ID NO: 4 (i.e., ATCTCTAGTCTGATGAGGCCGAAAGGCCGAAACAAGAGTC). While SEQ ID NO: 3 includes a ribozyme that is targeted to the +115 site (i.e., in units of count in the direction from the start)

transcription) of the wild-type U5 region, SEQ ID NO: 4 includes a ribozyme that is targeted to the +133 site of the wild-type U5 region.

Such a ribozyme is capable of cleaving within the wild-type HIV genome (or its transcripts) but not in the vector genome (or its transcripts), as long as the U5 sequences of the vector are modified by in vitro site directed mutagenesis as known in the art and described in Example 1. These vector sequences are particularly preferably modified such that the vector comprises a sequence selected from the group consisting of SEQ ID NO: 2 (i.e., GTGTGCCCACCTGTTGTGTGACTCTGGCAGCTAGAGAAC), SEQ ID NO: 5 (i.e., GTGTGCCCGCCTGTTGTGTGACTCTGGTAACTAGAGCCTCGTATA SEQ ID NO: 14, wherein at least one N is mutated, SEQ ID NO: 15 and SEQ ID NO: 16. In the form of RNA, the vector preferably comprises a sequence encoded by a sequence selected from the group consisting of: GTGTGCCCGTCTGTTGTGTGACTCTGGCAACTAGAGATC consists of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 14, wherein at least one N is mutated, SEQ ID NO: 15 and SEQ ID NO: 16. Conversely, wild type HIV includes a US sequence encoded by SEQ ID NO : 1 (ie GTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATC). These modifications as a whole and comparison to the wild-type sequence of U5 (in the form of DNA) are shown in Figure 2.

In addition, other ribozymes targeting other regions of the viral, particularly the HIV genome, may be used alone or in combination. For example, a ribozyme can cleave within other RNA sequences required for virus replication, e.g., within a reverse transcriptase, protease, or transactivating protein, within Rev, or within other indispensable sequences as described. Preferably, the vector comprises multiple ribozymes, eg, targeted to multiple sites.

In these cases, the analogous sequences in the vector are modified by site directed mutagenesis or some other method known in the art to produce a vector that is resistant to this ribozyme cleavage.

Preferably, if the vector is a human immunodeficiency virus, the vector lacks a tat gene and a 5 'splice site for the tat gene, and instead contains a triple anti-Tat ribozyme cassette in which the catalytic domain of each ribozyme from the triple cassette cleaves another site of the viral nucleic acid molecule. wild type human immunodeficiency, especially another site within tat. Preferably, the catalytic domain of each ribozyme cleaves a nucleotide sequence in a region of a wild-type human immunodeficiency nucleic acid molecule for which there is no ribozyme-sensitive counterpart in the vector itself.

Another embodiment of ribozymes, as a genetic antiviral agent, is obtained in ribozymes that target sequences not only according to the Watson and Crick base pairing principles, but also according to the varying base pairing of G-U. Previous studies have shown that fluctuating GU pairing in double-stranded RNA has similar features to Watson and Crick base pairing. Considering the high mutation rate, even of highly conserved regions, of HIV that gives rise to different strains of HIV, the ability to construct a ribozyme that targets multiple strains provides a dramatic advantage to the present invention. For example, the highly conserved target region in the tat gene, which can be used as a target sequence for ribozymes of the invention, contains either "G" or "A" in 40 well-defined HIV-1 strains. Thus, a ribozyme targeting this region can be designed to contain a "U" instead of a "C" in the appropriate targeted sequence to allow pairing with either the "G" or "A" residue. This can be compared to using "C" in the targeted sequence that would reduce the ability of the ribozyme to target HIV-1 strains that contain "A" at the corresponding position. This approach, using "U" wherever there is a G, A variability at the target position for ribozyme recognition, greatly increases the convenience of the ribozymes of the invention.

Another embodiment of the invention in practice is the use of a ribozyme cassette (which contains more than one tandem-bound ribozyme), degenerate such that it is not a directly repeat sequence in the vector. The presence of degenerate sequences prevents the deletion of repeat sequences that is observed in retro viral vectors and other nucleic acids. Indeed, a ribozyme cassette, consisting of tandem-aligned directly repeated sequences, can be deleted during multiple cycles of replication. Degenerate ribozyme sequences can be used to overcome this problem, because, for example, sequences in the catalytic domain of a hammerhead ribozyme molecule can be substituted for other nucleotides without significant loss of activity by varying base pairing. Mutagenesis of hammerhead ribozymes has been performed and sites that can be degenerated have been identified (Rufiner DE, Stormo GD, Uhlenbeck OC. Sequence requirements for hammerhead RNA self-cleaving reaction. Biochemistry. 1990 Nov 27; 29 (47): 10695- 10702). Another way of reducing the likelihood of deletion is to direct individual ribozymes from the cassette to different target sites of the target RNA. Thus, there will be no direct repeat sequences in the cassette.

A " transdominant " or " dominant negative " nucleic acid sequence transmits the phenotype encoded therein even in the presence of a wild-type (wt) sequence. An example is the above discussion of truncated CCR5 protein. Other examples include any mutant retro viral or lentiviral sequence that transmit a transdominant phenotype. Examples include the rev and gag genes. Examples of uses to obtain a selective advantage, as well as to inhibit infectious wild-type virus, are transdominant gag sequences that have been shown to inhibit HIV-1 replication, probably through interference with capsid construction. This

the sequence is then preferably used in conjunction with a non-HIV-1 based retroviral vector, allowing its replication and packaging without being inhibited by this transdominant sequence. For example, a retroviral vector that contains an HIV-2 gag and a pol sequence may include a transdominant HIV-1 gag sequence. This vector can be replicated and enveloped by complementation with an appropriate HIV-2 helper vector construct. Once placed in a cell exposed to HIV-1 infection, the transdominant gag sequence after HIV-1 infection is available for expression to block HIV-1 virus mobilization. However, the gag and pol genes encoded by the HIV-2 retroviral vector are still available for encapsulation and mobilization of the vector.

2. A nucleic acid sequence whose presence confers a selective advantage over cells infected or uninfected with a vector comprising the sequence over cells infected with wild-type virus.

A nucleic acid sequence that confers a selective advantage on a cell comprising a vector comprising this sequence over a cell containing or not containing a wild-type virus strain (i.e., which is absent) is preferably any sequence that allows a cell containing the vector to survive and multiply viral particles (i.e., crHIV viral particles) as compared to a cell containing a wild-type virus or a cell lacking this nucleic acid sequence. Such sequences include, but are not limited to, any sequence that allows a cell or vector contained therein to avoid destruction of sequences that promote cell survival, sequences that induce apoptosis, sequences that facilitate protein production, or sequences that support immune function or targeting.

For example, such a nucleic acid sequence contained in a vector preferably encodes broad spectrum drug resistance genes (see, eg, Ueda et al., Biochem. Biophys. Res. Commun., 141, 956-962, (1986); Ueda et al., 262,505-508). (1987) and Ueda et al., PNAS, 83, 3004-3997 (1987)). In the presence of an added cytotoxic drug (e.g., as used for cancer chemotherapy), this allows the cell containing the vector to survive, while the cell containing the wild-type virus, such as HIV, does not survive. Such cytotoxic drugs include, but are not limited to, actinomycin D, vinblastine sulfate, vincristine sulfate, BCNU (with or without BG conditioning), daunomycin, adriamycin, VP-16, and AMS A.

Another example of this nucleic acid sequence is any sequence variant of O ⁶ methylguanine-DNA-methyltransferase (MGMT). MGMT can be used to protect hematopoietic cell precursors from the toxicity of alkylating agents. Wild type MGMT is inhibited by O ⁶ -benzylguanidine (BG), which potentiates the toxicity of alkylating agents. By · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · The invention can be delivered to any cell of MGMT variants that are resistant to BG-mediated activation and capable of protecting against alkylating agents. One example of such a variant is G156A MGMT.

For example, hematopoietic precursor cells transduced with this variant of the present invention may be selected following administration of BG and O-guanine methylating or chlorethylating agents. Examples of such methylating agents for use in the present invention include, but are not limited to, clinically relevant temozolomide, nitrosoureas, tetrazines, triazines, dacabazine, temozolomide, streptozotocin, procarbazine. Examples of chlorethylating agents include, but are not limited to, BCNU (1,3-bis (2-chloroethyl) -1-nitrosourea), CCNU (3-cyclohexyl-1-chloroethylnitrosourea), ACNU (1- (4-amino) 2-methyl-5-pyrimidinyl) methyl-3- (2-chloro) -3-nitrosomethylurea) and MeCCNU (1- (2-chloroethyl) -3- (4-methylcyclohexyl) -1-nitrosourea) The alkylating agent is preferably BCNU.

It has been found that BCNU produces a permanent covalent interchain DNA lesion in both resting and cycling cells. These lesions are cytotoxic to cells during DNA replication. MGMT is the primary mechanism of DNA repair of BCNU lesions. Due to its toxicity to resting cells, selection by MGMT variants supplied by the lentiviral vectors of the invention can also allow selection of versatile hematopoietic stem cells (HSCs), which are predominantly in the Go cell cycle phase but which alternately cycle to produce more stem cells and blood cell precursors. Stem cells are generally resistant to retro viral transduction, but can be transduced with retro viral vectors only if they are not in the Go phase of the cell cycle. Similar to oncopetroviruses, the lentiviral vectors of the invention can be used to transduce hematopoietic stem cells, including detectable hematopoietic ELTC-IC and NOD-SCID or SCID-hu passaged cells. However, unlike oncetretroviruses, the cytokine conditions necessary for stem cell transduction without differentiation are likely to favor lentiviral vectors because lentiviruses (eg, HIV) can infect cells that do not actively divide at the time of infection. HSC selection is not possible with other strategies that select cycling hematopoietic cell precursors. Thus, selection mediated by MGMT variants and lentiviral vectors of the invention can increase the incidence of transduced, versatile HSCs without requiring prolonged drug administration for selection.

The above discussion can be viewed in the context of hemetopoietic cells in vitro and ex vivo as well as in vivo. Thus, in addition to transducing cells with MGMT variants in vivo, hematopoietic cells may be transduced ex vivo with subsequent drug treatment either ex vivo.

4 4 4 4

4 4

4 4

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or in vivo. These ex vivo treated cells may then be infused into the subject, optionally as part of a bone marrow repopulation with transduced cells.

Prior to such administration, the alkylating agent BCNU may also be used generally to reduce HIV-infected CD4 + cells in an HIV-positive subject. As mentioned above, BCNU (after depletion of endogenous MGMT) produces cytotoxic lesions in both resting and proliferating cells. These lesions are formed without a forced loss of MGMT if the dose of the drug exceeds the level of functional AGT protein. Repeated systemic treatment with BCNU causes cumulative myelosuppression and pancytopenia. Among lymphoid cells, CD4 + cells appear remarkably sensitive to BCNU. In subjects infected with retroviruses, such as patients with HIV infection, administration of BCNU, even at low doses, when removal of myelosuppression is desired, will reduce the overall population of CD4 + cells and thereby reduce viral load. This approach may be followed by infusion of hematopoietic precursors as discussed above.

Since BCNU can act in a "stealthy" manner and produce cytotoxic lesions even in resting cells, this approach has the additional advantage of reducing the need for repeated therapeutic interventions. This approach may, of course, be associated with the use of BG to enhance the effect of BCNU. Alternatively, this approach can be used in conjunction with the transfer of variants of MGMTs by the vectors of the invention to protect transduced CD4 + cells. The latter method may be either independent or in conjunction with the treatment of hematopoietic cells as mentioned above. Thus, a preferred embodiment of the invention is the expression of a mutant MGMT (second nucleic acid sequence) in a vector comprising an anti-HIV antisense sequence or ribozyme (first nucleic acid sequence) such that the vector provides both a selective advantage over wild-type HIV infected cells and a selective advantage over wild type HIV virus. Another preferred embodiment is that the vector expresses the MGMT gene (or second nucleic acid sequence) outside the HIV-LTR promoter, as a spliced mRNA.

Finally, although the vectors express anti-HIV ribozymes or antisense sequences, the invention is not so limited, as one skilled in the art can readily insert any inhibitory nucleotide sequence into the vector for a particular therapeutic, prophylactic or biological effect. A preferred method for expressing the inhibitory sequence is to include it in the U1 snRNA / promoter cassette as described in Dietz (US Patent 5,814,500).

It may also be desirable if the nucleic acid sequence comprises a sequence selected from the group consisting of a sequence (or a sequence that encodes) the mutated (ie, mutant) protease and a sequence (or a sequence that encodes) the mutated (ie, mutant) reverse transcriptase . Preferably, the mutated reverse transcriptase is constructed to be resistant to φφ · · · · · ·

ΦΦΦΦ • φ • ΦΦΦ

against nucleoside and non-nucleoside reverse transcriptase inhibitors, and the mutated protease is constructed to be resistant to commonly used protease inhibitors.

Administration of these protease inhibitors or reverse transcriptase to the host in conjunction with the vector is used to select cells that produce the vector as opposed to cells that produce the wild-type virus. Similarly, this approach is modified for use with any drug that inhibits viral replication, so that the virus can be mutated to avoid inhibition. Therefore, for the treatment of HIV, the selective nucleic acid sequence built into the vector preferably includes mutated HIV sequences. However, it is optimal if these sequences do not prevent superinfection with wild-type HIV.

Preferably, the vector is one of the vectors mentioned above, in particular the improved conditionally replicating vectors shown in Figures 1A to 1K. Of course, when used according to the method of the invention, GFP coding sequences can be deleted or replaced by other sequences. GFP coding sequences are present in these exemplary embodiments of vectors as a marker or indicator of the presence of the vector or gene expression from the vector.

Preferred vectors of the invention may comprise various elements described herein. In particular, lentiviral vectors may lack a tat gene and contain at least one antisense sequence. In addition, the vectors may comprise a sequence such as the central polypyrimidine region of HIV or a larger nucleic acid fragment containing it.

Optimally, the vector is compatible with the cell into which it is introduced, e.g., is capable of eliciting expression of the vector encoded nucleic acid sequences on the cell. Desirably, the vector comprises an origin of replication functional in the cell. When the nucleic acid sequence is transferred in the form of its DNA coding sequence (e.g., as opposed to the form of a complete gene containing its own promoter), it is optimal that the vector also comprises a promoter capable of directing expression of the coding sequence and operably linked to the coding sequence. sequence. A coding sequence is "operably linked" to a promoter (eg, if the coding sequence and the promoter together form a native or recombinant gene) if the promoter is capable of directing the transcription of that coding sequence.

In the recombinant vector of the invention, preferably all intrinsic transcription sites (e.g., initiation and termination signals), translation (e.g., ribozyme entry site or binding site, etc.), process signals (e.g., donor or acceptor splice sites, if necessary, and polyadenylation signals), translocation sites, assembly, integration, ribonuclease complex entry, stability and translocation elements, in cis or trans position, arranged appropriately transcribed (and / or translated, if desired) in cells, • 0000 00 0000 ·· 000 ·

0000 00 0 · 00000 00 0 0

000 ·· · 0 0 0 0 • 00 · 0 ·· ··· 0 · 00 the vector is loaded. Influencing these signals to ensure adequate expression in host cells is within the skill of the art.

Preferably, the vector comprises a Pol sequence that enhances the efficiency of stable transduction, defined as integrated copies of the vector in the genome of a host cell. The sequence previously identified by Zenna et al. (Cell 101: 173-185 (2000)) as a central DNA flank (a 178 base pair fragment from positions 4793 to 4971 on pLAI3, corresponding to positions 4757 to 4935 on pNL4-3) where it was reported to increase transduction efficiency was found is not sufficient to significantly increase the efficiency of stable transduction. The present invention includes the discovery that while this small fragment is not sufficient to increase the efficiency of stable transduction, a larger 545 base pair fragment (positions 4551 to 5096 in pNL4-3) or containing even larger fragments as described in US Patent 5,885,806 are capable of enhancing stable transduction as part of the present invention. Increased efficiency of stable transduction was detected by GFP expression and FACS analysis and Taqman analysis of integrated copies of the vector genome.

Other possibilities for increasing transduction efficiency are described in co-pending U.S. Patent Application Serial No. (to be assigned) filed August 31, 2000, under Representative No. 397272000400, which is hereby incorporated by reference in its entirety.

The viral vectors used in the present invention may also result from the generation of "pseudotypes" when co-infection of a cell by different viruses produces virion progeny comprising the genome of one virus encapsulated in the outer layer containing one or more envelope proteins of another virus. This phenomenon is used to coat viral vectors of interest into a "pseudotyped" virion by co-transfection or co-infection of the envelope cell with the viral vector of interest and genetic material encoding at least one second virus envelope protein or cell surface molecule; see US Patent 5,512,421. These mixed viruses can be neutralized by antisera against one or more heterologous envelope proteins used. One pseudotyping commonly used virus is the vesicular stomatitis virus (VSV), a rhabdovirus. The use of pseudotyping extends the host cell range for the virus by incorporating elements of the virus entry mechanism into the cell used in the heterologous viruses used. Pseudotyping of viral vectors and VSV for use in the present invention results in viral particles comprising the viral vector nucleic acid encapsulated in a nucleocapsid that is surrounded by a membrane comprising the VSV G protein. Preferably, the nucleocapsid comprises proteins that are normally associated with the viral vector. The enveloping membrane containing the VSV G protein forms part of the viral particle upon its exit from the cell that was used to envelop the viral vector. Examples of enveloping cells are described in US Patent 5,739,018. In a preferred embodiment of the invention, the viral particle is 9 9 9 9 999 9 9 9

9 9 9 9 9

9 9 9 9 9 9 9

999 99 99 · 99 99 99 derived from HIV and pseudotyped with the VSV G protein. Pseudotypic viral particles containing the VSV G protein can infect multiple cell types with greater efficacy than amphotropic viral vectors. The host cell range includes mammalian and non-mammalian species such as humans, rodents, fish, amphibians and insects.

Preferably, the vector also comprises some means by which the vector or a subcloned sequence can be identified and selected. The identification and / or selection of the vector is accomplished using a variety of approaches known to those skilled in the art. For example, vectors that contain certain genes or coding sequences are preferably identified by hybridization, the presence or absence of so-called "marker" gene functions, encoded by the marker genes present in these vectors, and / or the expression of certain sequences. In the first approach, the presence of a particular sequence in the vector is identified by hybridization (eg, DNA-DNA hybridization) using probes containing sequences that are homologous to the relevant sequence. In a second approach, a vector / host recombinant system is identified and selected based on the presence or absence of a particular marker gene function, such as antibiotic resistance, thymidine kinase activity, and the like, elicited by particular genes encoding these functions present in the vector. In the third approach, vectors are identified by analyzing for a particular gene product encoded by the vector. These assays are based on the physical, immunological, or functional properties of the gene product.

Accordingly, the present invention also provides a vector which, if DNA is, comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2,4, 5, 6, 15, and 16, and which, when RNA is comprising a nucleotide sequence encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4,5,6.

The present invention further provides a method of combining a vector that is derived from a wild-type human immunodeficiency virus and which is capable of replicating only in a host cell capable of replicating the vector with a ribozyme. A ribozyme that is contained in or encoded by the vector cleaves wild-type nucleic acid. of human immunodeficiency virus, but not the vector itself and its transcripts, if any. The method comprises obtaining a vector which is derived from a wild-type human immunodeficiency virus and which is capable of replicating only in a host cell that permits replication of the vector, and introducing a nucleic acid sequence into the vector, which sequence comprises or encodes a ribozyme whose catalytic the domain cleaves wild-type human immunodeficiency virus nucleic acid, but not the vector itself and its transcripts, if any. In this procedure, a nucleotide sequence containing or encoding U5 may be deleted from the vector

9999 • 9

9 99

9999 · wild-type human immunodeficiency virus sequence and replaced by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 5, 6,14, wherein at least one N is mutated, 15 and 16 if the vector is DNA, and a nucleotide sequence encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 5,6,14, wherein at least one N is mutated, 15 and 16 when the vector is an RNA. Preferably, the vector replicates in a host cell that allows said vector to replicate more than once.

Also provided by the present invention is a method of modifying a vector. The method comprises obtaining a vector and introducing into said vector a nucleotide sequence selected from the group consisting of the DNA sequences of SEQ ID NOs: 2,3,4, 5,6,14, wherein at least one N is mutated and 15 and 16 are mutated. if the vector is DNA, and a nucleotide sequence encoded by a nucleotide sequence that is selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 15, and 16 if the vector is RNA.

Further, the present invention provides a method of propagating and selectively enveloping a conditionally replicating vector without using a enveloping cell line. The method comprises contacting a conditionally replicating vector with a cell which is capable of being infected by another vector that is of the same type of vector as the conditionally replicating vector and that differs from the conditionally replicating vector by being wild type with respect to competence to replicate; subsequently contacting said cell with said second vector and then culturing said cell under conditions conducive to propagation of the conditionally replicating vector. The helper vector discussed above and in more detail below is one such complementary vector.

Also provided is an isolated and purified nucleic acid molecule selected from the group consisting of a DNA molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 5,6,14, wherein at least one N is mutated, 15 and 16, and RNA molecules comprising a nucleotide sequence encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2,6,15 and 16.

Methods of use

The above-described vectors are preferably introduced into a host cell for the prophylactic and therapeutic treatment of a viral infection, for ease of maintenance of the vector, as well as for other reasons. Accordingly, the present invention provides a host cell comprising a vector of the invention. Isolation of the host cell and / or maintenance of these cells, or cell lines derived therefrom, in culture has become a routine matter in which one of ordinary skill in the art is familiar.

· Μ · * · <· 99 9999

9 9 9 9 9

9 9,999 9 9 9

9 9 9 9 9

9 9 9 9 9

99 99 999

In particular, a conditionally replicating vector or preferably a lenti viral vector as described above is preferably used in the prophylactic and therapeutic treatment of a viral infection, preferably when the infection is a wild-type virus, preferably a wild-type RNA virus, more preferably wild type retrovirus and optimally wild type. HIV.

The method comprises contacting a host cell capable of being infected with a wild-type virus with a conditionally replicating vector that is capable of replicating only in a host cell that allows replication of the vector whose presence, transcription or translation inhibits the replication of the wild-type virus strain in the host cell. Desirably, the vector replicates more than once and comprises at least one nucleic acid sequence whose existence (ie, presence, transcription or translation) provides the vector in a host cell with a selective advantage over a wild-type virus strain, which is optimally the strain from which this vector was derived.

Accordingly, the nucleic acid sequence preferably comprises a nucleotide sequence that contains or encodes a genetic antiviral agent that adversely affects the replication and / or expression of a virus other than said vector. Desirably, the genetic antiviral agent is selected from the group consisting of an antisense molecule, a ribozyme, and an immunogen. Optimally, the genetic antiviral agent is a ribozyme whose catalytic domain preferably cleaves the 3 'nucleotide sequence of the NUH (i.e., particularly the GUC sequence). Optionally, the ribozyme is encoded, at least in part, by a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4. Desirably, the ribozyme cleaves in the wild-type region of the virus strain or its transcripts, but does not cleave in the region of the vector or its transcripts. This is preferably because this wild-type virus strain contains the sequence encoded by SEQ ID NO: 1, whereas the vector, if DNA is, comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 5,6, 14, wherein at least one N is mutated, 15 and 16, and, if RNA is, comprises a nucleotide sequence encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 5, 6,14, wherein at least one N mutant, 15 and 16.

This method is also preferably performed when the vector comprises at least one nucleic acid sequence whose existence (ie presence, transcription or translation) confers a selective advantage on a cell which is infected with the vector over a cell that is infected with a wild-type virus strain that optimally, it is the strain of the virus from which this vector was derived.

In this regard, the vector may comprise at least one nucleic acid sequence which confers a selective advantage on a cell which is infected with the virus and at least one nucleic acid sequence. 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 which gives a selective advantage to the vector over the wild-type virus strain corresponding to the virus from which the vector was derived.

Accordingly, the method is preferably performed when the nucleic acid sequence comprises a nucleotide sequence that encodes a broad spectrum drug resistance gene. Alternatively, the method is performed when the nucleic acid sequence comprises a nucleotide sequence encoding a mutated (mutant) protease and a nucleotide sequence encoding a mutated (mutant) reverse transcriptase, for example, when a viral infection to be treated prophylactically or therapeutically is caused by a retrovirus.

The method further preferably comprises administering to the host cell an agent selected from the group consisting of a cytotoxic drug, a protease inhibitor, and a reverse transcriptase inhibitor (i.e., in addition to administering the vector).

Accordingly, the vector may be used in accordance with the above method, not only for the therapeutic treatment of a viral infection, but also for protecting a potential host cell from a viral infection, i.e. a method of prophylactic treatment of a viral infection or "vaccination" against a virus of interest, RNA virus, in particular a retrovirus such as HIV. This method substantially inhibits the replication of the wild-type virus strain before the host cell contacts this wild-type virus strain. In this regard, the vector may contain or encode proteins that block superinfection with a wild-type virus. The method comprises contacting the host cell with a conditionally replicating vector as described above and a "helper expression vector", ie a viral genome that promotes replication of the "vector" in an uninfected host. The conditionally replicating vector contains a selective advantage in packaging and / or propagation. In addition, the vector may, for example, comprise a sequence that enhances cell survival, promotes virus production, induces apoptosis, facilitates protein production, and / or promotes immune function and / or targeting. A "helper expression vector" construct is any expression vector that compensates for the "vector" inability to replicate. These helper expression vectors are conventional and are readily assembled by one of ordinary skill in the art. This helper expression vector can either be packaged into virions as a vector or expressed without the need for packaging. Because such a "vector" has a selective advantage in packaging and / or propagation, this system provides a safe means to achieve high virus replication without the possible pathogenic effects that a live attenuated virus could potentially cause. In addition, the vector may be mixed with a non-specific adjuvant to enhance immunogenicity. These adjuvants are known to those skilled in the art and include, without limitation, Freund's complete or incomplete

9949 · 9 9 9 <9 9 9 • 9 9 9 9 9

9: 9 9 4 4 9 4 9 ·· «9 · 9 · 49 99 adjuvants, emulsions consisting of bacterial and mycobacterial cell wall components and the like.

Conditionally replicating viral vectors or preferably lentiviral vectors of the invention may also be used for immunotherapy in the treatment of viral infection or for the treatment of, for example, oncogenic disorders. Dendritic cells or precursors thereof (eg, but not limited to CD34 + hematopoietic cells or blood monocytes), as well as other antigen-presenting cells, can be transduced with a vector that expresses HIV proteins or a protein sequence that contains major epitopes, using wherein the host implements an immune response to a foreign agent such as a virus. Such protein epitopes for HIV are described in the &quot; HIV molecular immunology database &quot;, 1998, National Institute of Allergy and Infectious Diseases, Maryland &amp; Los Alamos National Laboratory, New Mexico, which is hereby incorporated in its entirety. A preferred embodiment of the HIV epitope protein is an integrated or composite CTL sequence (or fragment thereof) shown in Example 14 below. Other epitopes that can be expressed similarly are derived from other viruses, bacteria, fungi, parasites, tumor cells, and genetically engineered cells. . More than one epitope may be linked to a protein sequence so that non-immunogenic sites are excluded to create a vector with enhanced safety because the protein coding sequences are strictly discontinuous. A preferred embodiment of the discontinuous sequence is a degenerate discontinuous sequence (with or without corresponding linker amino acid sequences to stabilize the protein structure, if necessary), encoding immunologically relevant epitopes. This use of degenerate sequences reduces the risk of recombination. Another preferred embodiment is to express the described protein sequence (or fragment thereof) using a retroviral LTR-driven attached message, such as HIVLTR.

However, the use of retro viruses in the treatment of human infections and diseases is associated with the risk of producing a replication competent virus (RCV). Homologous recombination between the helper vector and the conditionally replicating vector appears to be one of the main directions of RCV generation. Therefore, the present invention allows the modification of the helper vector construct to minimize or eliminate the possibility of homologous recombination. The invention includes any modification which serves to reduce the likelihood of dimerization, co-packaging and / or recombination of the helper vector and the conditionally replicating vector.

One embodiment of the invention consists in inserting one or more anti-vector ribozymes or an antisense sequence (optionally in the form of a cassette defined as at least two such tandem linked sequences with respect to ribozymes and defined as at least two such tandem linked sequences targeted to discontinuous regions) of the target vector with respect to antisense sequences) to the 3 'end of the coding sequences of the helper gene, but 5' to the transcription termination site. Although nonspecific packaging of helper genomes into the vector may occur, it is likely that such packaging is vector-dependent. Thus, many strategies can be used to reduce the encapsulation of the helper vector into vector particles that would otherwise be the first step in the possible generation of RCV.

A preferred helper construct comprises an anti-vector ribozyme or antisense molecule at the 3 'end of the structural protein coding sequence or envelope coding sequence (homologous or heterologous). A more preferred helper construct comprises, at the 3 'end, a structural protein coding sequence and an envelope coding sequence, an anti-vector ribozyme or an antisense molecule. If the nucleic acid sequence is an antisense molecule, it may also function intracellularly to cleave the co-localized double-stranded helper vector molecules by cellular nucleases. If the helper is enveloped by the vector RNA in the form of viral particles, then these molecules are unable to undergo complete reverse transcription to form RCV.

In another preferred embodiment, a sequence is supported on the helper construct that promotes a different location or location of the helper nucleic acid remote from the nucleic acid of the vector. An example, but not limited to, is the incorporation of heterologous intron and poles of A sequences into helper constructs, as shown, for example, in Figure 6. These sequences may facilitate different placement of vector and helper nucleic acid at different cellular and subcellular positions. The titer of the vector produced by this helper modification is preferably not more than 100 fold, more preferably not more than 10 fold, and most preferably not affected.

Figure 7 shows that the presence of a single ribozyme (pVirPac1.2RzIn) or a ribozyme and intron designed to affect helper RNA cellular transfer (pVirPac1.2RzIn) had no significant effect on vector titer. Figure 12 shows that PCR analysis of titrated samples on co-coated helper constructs showed a decrease in co-coating in the absence of ribozymes compared to high co-coating in the absence of ribozymes. The presence of two ribozymes completely prevented the helper vector from co-enveloping the vector virus particles. Thus, the present invention encompasses an effective means of using a two plasmid envelope system for both producing high vector titers and improving the safety of these vectors by reducing or eliminating co-coating of the helper vector.

These data suggest that helper enveloping into virions is not random, but is vector dependent. An alternative preferred embodiment to avoid co-enveloping helper nucleic acids with vector nucleic acids is the degeneration of the helper construct in regions that are important for joining the helper and vector sequences. This approach can be further modified by complete degeneration of the helper sequence to prevent co-packaging of the helper and vector sequences.

For example, the nucleotide sequence of the helper vector may be degenerated either partially or completely. While such degeneration significantly reduces or eliminates the likelihood of homologous pairing and recombination of the helper vector and the conditionally replicating vector, it does not affect the ability of the helper vector to encode protein products necessary for virus replication and packaging. This degeneration may be directed to any possible gene and an open reading frame (ORF) that is homologous between the helper and the conditionally replicating vector, or more specifically targeted to particular genes or within particular genes or ORFs in the helper vector. It should be noted that complete degeneration is not necessary to reduce the likelihood of homologous recombination, since the absence of sequence homology greater than 18 nucleotides in length should be sufficient. Degeneration to a level where there is no more than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 nucleotides identical between the sequences will suppress or prevent recombination. if desired, degenerate using codons that have preferential expression in human or certain cell types.

By way of example, the gag, rev and / or tat coding sequence may be degenerated either in whole or in part. Other examples include degeneration of the first 42 nucleotides of the gag sequence, the first 208 nucleotides of the rev sequence, the last 183 nucleotides of the tat sequence, and the last 545 nucleotides of the Pol sequence. Examples of helper vectors with such degeneration are pVirPac1.2, pVirPac1.2Rz, pVirPac1.2Rz2 and pVirPac1.2RzIn or derivatives thereof. Of course, the nucleotide sequence encoding any other gene product may also be degenerate according to the invention, thereby reducing, minimizing or eliminating the likelihood of homologous recombination.

An alternative embodiment of the invention is to incorporate into the helper vector an element that intervenes and degrades the conditionally replicating vector when co-location, co-packaging or other joint pairing of the two vectors occurs. An example of such an element is a ribozyme. Any ribozyme that selectively affects a conditionally replicating vector is within the scope of the invention. Multiple ribozymes may also be used, such as the double or triple ribozyme cassettes described herein. For example, the ribozyme may target the U5 region (eg, the U5 region of HIV-1, HIV-2, or other retrovirus used in a conditionally replicating vector). Cleavage of the conditionally replicating vector prevents RCV from recombining with the conditionally replicating vector, which can occur if: · · · ·

the two vectors are packaged together. Examples of ribozyme-containing helper vectors are pVIRPAC-1.1Rz and pVIRPAC-1.2Rz2, as shown in Figure 6.

Yet another embodiment of the present invention is the substitution of heterologous RRE, including any retro viral RRE, but preferably other lenti viral RRE, into a helper vector, such as substitution of HUIV-2 RRE for HIV-1 RRE, CTE or PRE. This approach contributes to reducing, minimizing or eliminating the possibility of homologous recombination based on different RREs having different sequences. A further advantage of such substitution according to the invention is the surprising and unexpected increase in the production of the conditionally replicating vector, up to about 5-fold. Without wishing to be bound by theory, the presence of different RREs between the helper vector and the conditionally replicating vector may be bound to one or two different cellular factors, which may be limited in amount. Thus, the use of different RREs can reduce competition between the two vectors by limited cellular factors. Examples of helper vectors containing the HIV-2 RRE element for enveloping an HIV-1-based retroviral vector are pVIRP AC-1.2 and pVIRPAC-1.2Rz2 as shown in Figure 6.

In yet another embodiment, a nucleotide sequence encoding a heterologous env protein, such as VSV-G from vesicular stomatitis virus, rabies G protein, GaLV, alphavirus E1 / 2 glycoprotein, or RDI 14, fox endogenous env protein, is incorporated into the hepler vector. virus. This allows the conditionally replicating vector to be enveloped in particles with heterologous env proteins. In addition, a ligand may optionally be inserted into the envelope protein to promote stimulation of the target cell during transduction, if desired. Since modification of the envelope protein may impair its binding ability, it is a preferred embodiment to express both a modified ligand containing the chimeric envelope protein and an unmodified, pseudotyped envelope protein during production. Thus, both types of envelope proteins may be on the cell surface, one to stimulate the target cell and the other to mediate binding and entry. A preferred pseudotyped lenti viral vector that also expresses the chimeric receptor for cellular stimulation is the VSV-G envelope protein and the chimeric VSV-G envelope protein. A more preferred chimeric envelope protein is the VSV-G / RD114 chimeric envelope protein (see Figures 14A and 14B). Other preferred proteins (or fragments thereof) for the formation of VSV-G chimeras include notch, delta, FLT-3 ligand, TPO, Kit ligand, CD3 binding ligand, CD28 binding ligand, and GM-CSF binding ligand.

Optionally, the sequence encoding the heterologous env protein may be operably linked to a second inducible promoter to provide greater env protein delivery for viral envelope. This approach may further increase virus production, particularly under conditions where env protein production is a rate limiting factor. This heterologous env protein coding sequence can either be contained in the same helper vector as the second complementary viral protein coding sequence or on another vector. Optionally, it may also be incorporated into a producing host cell or cell line.

Another embodiment of the present invention is to selectively locate a splice donor and acceptor site on a helper vector in such a way that selective splicing of certain viral components serves to minimize or eliminate the possibility of homologous recombination with conditional replication. For example, these splice sites may be positioned to allow RRE deletion and / or wrapping of dimerization signals as a result of splicing. Thus, the continuity of whether the nucleic acids are expressed can be controlled by placing splicing sites in the vector. For example, splice sites may be located distal to the ribozyme or antisense sequence such that ribozymes or antisense sequences are incorporated, for example, in uncut, but not spliced, RNA. Alternatively, these ribozymes or antisense sequences may be located downstream of the splice site if it is intended to express ribozymes or antisense molecules from all mRNA molecules and not just a particular subtype.

When used in accordance with the above method as a prophylactic treatment of a viral infection, the vector may encode an antigen of a protein that is not encoded by a wild-type virus, such as a mutant viral protein or a non-viral protein. Accordingly, the antigen encoded by the vector may be, for example, of bacterial origin or from a cancer cell. In addition, the conditionally replicating viral vector or preferably the lentiviral vector may also encode an MHC gene for proper presentation of the antigen to the host immune system. Thus, these vectors can be used to facilitate a sustained immunological response against a variety of potential pathogens and / or endogenous proteins (eg, tumor-specific antigen) that are selectively expressed in abnormal cells.

The above example does not limit the use of the invention to deliver antigen to dendritic cells for immunotherapy. Conversely, the invention provides means and methods for delivering any gene of interest and its expression in a cell of any desired type. In addition, the invention also guarantees the delivery and expression of multiple genes by a single vector. Multiple gene expression may be accomplished by any suitable gene expression strategy. A non-limiting example is the use of internal ribosomal entry sites (IRES), which can be located distal to the first gene to be expressed. A preferred example is the expression of the gene of interest into an IRES sequence that is distal to the variant MGMT gene. A more preferred embodiment of the vector expresses a gene of interest and an IRES linked gene of interest from an unmodified or modified HIV-LTR promoter outside the spliced messenger sequence. Another preferred vector expresses two genes of interest

from an unmodified or modified HIV-LTR promoter which, if modified, is modified in sequences that bind transcription factors. Of course, any unmodified or modified lentiviral LTR may be used in addition to the HIV-LTR.

Another non-limiting example is the expression of the gene of interest from HIV-LTR using HIV-derived splice sites. For example, one gene of interest may be expressed from a Nef splice acceptor site, while the other gene of interest may be expressed from a Tat splice acceptor site. In this way, two genes can be expressed without the need for an IRES element. Any splice site can be used, including the Tat, Rev, Vif, Vpr, Vpu, Nef, and Env gene sites. The Env protein is expressed from a single spliced messenger sequence, so expression of the gene of interest via this splice site may require Rev if significant expression of the gene of interest is required. In addition, if desired, expression from HIV-LTR can be made to be dependent on Tat and Rev.

In addition, a helper viral expression vector (also referred to herein as a "helper") can be engineered to express only certain cell types (eg, stem cells, specialized antigen presentation cells, and tumor cells) by adding or deleting a particular genetic element. factor (either in the vector or in the helper virus expression construct), allowing for cell-specific replication and propagation of the vector. Thus, the vector is still propagated by complementation with the helper virus construct, but this spread is cell-specific, depending on whether a particular genetic element / factor is added or omitted in the vector or in the helper virus expression construct. This can be used alone or in conjunction with any of the above strategies.

For example, a conditionally replicating HIV vector can be constructed for specific replication in macrophages instead of in T-cells. A vector that produces Tat-defective HIV (the vector encodes other HIV proteins, but these are not expressed due to the absence of Tat transcriptional activator) can encode a ribozyme that cleaves wild-type HIV, but not conditionally replicating HIV RNA. The helper expression vector for this vector can encode a tat gene expressed from a macrophage-specific promoter. Thus, crHIV will conditionally replicate only in macrophage cells, while it will not be able to replicate in cells or other cell types.

Alternatively, the tat gene can be operably linked to a tumor-specific promoter; Thus, crHIV will then only replicate in CD4 tumor cells and not in normal CD4 cells. The genetic element / factor may also be a modification of the promoter sequence of a vector that is expressed only in certain cell types and not in other cell types, in accordance with the helper-virus expression construct.

In another embodiment, the envelope proteins of a helper expression construct or vector construct (if such constructs are designed to contain envelope proteins) may be modified such that the vector-virion will specifically infect certain cell types (eg, tumor cells) while not being able to infect other cell types (e.g., normal cells).

In yet another embodiment, an adenovirus that lacks one or more key factors for replication can be complemented using a helper construct that provides these factors, bound to a tumor-specific promoter. Thus, these factors that complement adenovirus replication can only be expressed in tumor cells, thereby allowing virus replication in tumor cells (expressing proteins required for cell killing), but not in normal cells.

In another preferred embodiment, the vector can express a negative selection gene for killing cells using O ⁶ -guanine alkylating agents such as, but not limited to, BCNU (or a functional analogue of BCNU) as a negative selection drug. A lentiviral vector expressing an antisense sequence or ribozyme targeted to the MGMT gene can be delivered to normal cells. At a later desired time, these cells may be treated with BCNU or an analogue thereof, either ex vivo or in vivo, to kill cells that have been transduced with the vector. In a preferred embodiment, normal cells are pro-phylactically transduced with an MGMT antisense expression vector and are later killed when they become abnormal. An example of this latter approach is when these transduced cells become cancer cells, which can then be killed by treating these cells with BCNU or a functional analogue thereof. Another preferred embodiment is to express the antiMGMT antisense sequence or ribozyme molecule from the U1 promoter and contained in the U1 snRNA. An even more preferred embodiment is to transduce the anti-MGMT lentiviral vector into a population of hematopoietic cells such as lymphocytes, stem cells and dendritic cells.

In another preferred embodiment, the normal cells are hematopoietic cells, preferably T cells, which are transduced by the vector prior to transplantation into an allogeneic host. These cells can be killed by treatment with BCNU or a functional analogue thereof if they become harmful to the host (e.g., if the host is defective against the graft).

In yet another embodiment, the same lentiviral vector experimenting with an anti-MGMF antisense sequence or ribozyme can be used to remove unwanted cells from pooled cell populations. A non-limiting example is in the case of bone marrow contaminated with cancer cells that is ready for transplantation. It has been observed that tumor cells transduce very efficiently at relatively low MOIs in one cycle of transduction (see Figure 13A). In contrast, normal cells, in particular, but not limited to CD34 + hematopoietic cells, are much more.

9 9 9 9 more difficult to effectively transduce and require multiple transduction cycles to achieve high efficiency (see Figure 13B). Thus, an attractive purification strategy is the transduction of contaminated bone marrow with a vector containing an anti-MGMT antisense sequence, using an MOI that will effectively transduce contaminating tumor cells but not normal cells. This example does not limit the scope of the invention either to its ex vivo use or only to cancer-related applications. Other applications include in vivo selective gene delivery, in particular, but not limited to, in the treatment of brain cancers and any other disease where there is a different transduction efficiency between diseased and normal cells that can be used for targeting according to the invention.

The present invention therefore provides a method of treating cancer, and in particular, treating T-cell leukemia. The "cancer treatment" of the invention comprises administering to a host of a further modified vector as described herein to elicit a therapeutic response. Such a response can be ascertained by observing a decrease in tumor growth and / or tumor regression. "Tumor growth" includes increasing tumor size and / or number of tumors. "Tumor regression" includes diminishing tumor mass.

The "cancer" of the present invention includes cancer, which is characterized by abnormal cell proliferation and absence of contact inhibition, as evidenced by tumor formation. The term includes cancer localized in tumors, as well as cancer not localized in tumors, such as cancer cells that spread from the tumor locally by invasion or systemic metastasis. Theoretically, any type of cancer may be the subject of treatment according to the invention. However, it is preferred that the cancer is of viral origin.

Finally, the above vectors can be directly used for in vivo gene therapy. Current gene therapy strategies suffer because they cannot mediate gene delivery to a large percentage of cells; only a certain percentage of cells are infected. This is particularly important in antitumor strategies where gene transduction into the entire tumor population is critical. Upon addition of the "vector" in collaboration with the "helper", transduced cells will immediately produce viral particles that can infect neighboring cells and thus allow high and possibly complete transduction efficiency. In one embodiment of the invention, a human retrovirus (which may be an HIV or retrotransposone element) can be delivered to a tissue (or in vitro cell) by a "helper" construct. Cells immediately containing this vector and helper will produce virus and envelop the vector conditionally into virions. These virions will be able to mediate high transduction efficiency of adjacent cells (since cell-cell contact is the most efficient way of transducing cells). Immediately transduced cells • ·· • 9,999

999 · 9 9 9 9 β may or may not die, depending on whether the vector / helper combination results in a cytolytic infection. In the case of retrotransposone, the helper may not contain structural proteins since normal or tumor cells may contain the protein / factor necessary for encapsulation into virions. In this case, the helper may be merely, but not limited to, a transactivating protein that activates the transcription of factors necessary for encapsulation of retrotransposone. In the case of HIV, other factors may be necessary, but not necessarily, to encapsulate the HIV genome in progeny virions for cell infection / transduction.

The vectors described above can also be used in biological or chemical weapon strategies. For example, a conditionally replicating vector may be delivered to an individual recently infected with a highly pathogenic virus or bacterium or chemical agent (eg, a toxin). This vector will interfere with the replication of the pathogenic virus as described above. However, the conditionally replicating vector can also be used for antibacterial or anti-chemical strategies in accordance with a helper expression vector ("helper").

For example, a conditionally replicating vector may secrete antibacterial or antitoxin antibodies after the helper allows its expression and propagation. This helper may, but is not necessarily, driven by an inducible promoter that guarantees its expression upon activation by bacteria, a cytokine (in response to a bacterial infection), an antibiotic (as in the case of tetracycline-inducible promoter systems) (Paulus et al., J. Virol) ., 70, 62-67 (1996)) or chemicals (e.g., toxin alone), therefore, this conditionally replicating vector will not merely selectively multiply with the help of a helper in response to an impending pathogen or toxin (as a result of helper activation), but will also secrete anti-pathogenic or antitoxin antibodies to inhibit the pathological effects of tumor antigens, pathogens or chemicals (eg, toxin). Therefore, any protein, factor, or genetic element that can either be transcribed can be inserted into the conditionally replicating vector. into mRNA and / or protein to inhibit the pathogenic response - in accordance with "h elper 'that promotes its selective propagation and expression (selective because helper products are expressed conditionally (for example, but not limited to, (a) an inducible promoter-factor in a tumor cell activates helper factor production, a toxin-sensitive sequence that expresses helper factor, or cytokine-sensitive promoter, induces helper factor production, (b) helper RNA / protein / factor is selectively stabilized in certain cells and not in others), and (c) indirect induction of a third-party gene that affects helper viral viral production protein, accompaniment, targeting, structure or other biological functions). These strategies can be used in transgenic plants and animals to protect them from pathogens. Similarly, 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 These strategies are used in transgenic systems to produce valuable heterologous proteins / factors.

In another embodiment of the method of the present invention, a cell line for drug / factor screening can be developed, for example, to determine which portion of the protein / factor is important for a particular function. A vector can be constructed to express the mutated protein of interest in a given cell line. However, the RNA encoding this mutated protein is engineered to be resistant to ribozymes, for example by inserting silent point mutations. However, expression of the wild-type protein is inhibited in the cell line. Vectors that express a ribozyme to this protein of interest can also be engineered to express a mutant test protein. When this vector is transduced into cells, most of the native RNA encoding the normal protein will be cleaved while the mutant test protein will be expressed. This method can be used with concomitant delivery and selection methods as a fast and powerful method for determining how a given protein works and how a given factor / drug interacts with that protein.

In yet another use of a vector for treating blood-borne diseases, the patient undergoes leukapharesis to extract sufficient white blood cells from the blood. After leukapharesis, these desired cells are isolated, transduced with the vector, and then expanded to the desired number of cells in culture. During the ex vivo multiplication of this desired cell type, the patient receives an infusion of antibodies against the cell surface proteins of the desired cell type, with the intention of breaking these cells over time, corresponding to the expansion of a sufficient amount of the desired cell type. The transduced cells are then returned to the patient by infusion to compensate for the in vivo antibody-induced cell loss.

A preferred embodiment of the above is isolating CD4 + T cells from the leukapharesised material and then transducing these cells with an HIV vector containing an anti-HIV antisense or ribozyme sequence for ex vivo extension. During ex vivo cell proliferation, the patient's endogenous CD4 + T cells are destroyed by administering, for example, anti-thymocyte globulin (ATG) to Pasteur Merieux Serums et Vaccins, Lyon, France, supplied by SangStat Medical Corporation, Menlo Park, CA, USA or Atgam (Upjohn Company, Kalamazoo, MI, USA), but any cytoreductive antibody may be used after prior screening, as will be apparent to those skilled in the art. Upon spreading, these transduced CD4 + T cells are returned to the patient by infusion. In a preferred embodiment, the CD4 negative cells obtained during the isolation of CD4 positive cells are captured, frozen and melted for infusion when the transduced CD4 positive cells are returned to the patient by infusion.

The above example does not limit the scope of the invention to either ATG or CD4 + T cells. Any cell type may be the target of using an antibody that binds cell surface protein.

4 • 440 ········

This cell is cytocidal. The antibody may be introduced exogenously into the patient or a second vector secreting the antibody may be introduced into the body. This vector may produce the antibody either constitutively (throughout the life of the cell) or transiently (for example, but not limited to an integrase minus vector) or inducibly (for example, but not limited to a tetracycline-inducible promoter system).

There are also many uses of the methods and vectors of the invention in vitro. For example, these vectors can be used to detect certain nuances of viral replication and ribozyme function. Similarly, ribozyme containing vectors can be used as diagnostic tools, for example, to detect mutations present in diseased cells, or to investigate genetic drift. These vectors may also be used to determine or confirm the function of a gene, whether this function is known in advance, unknown, or merely indicated or contemplated. The above discussion is by no means exhaustive as to the application of the invention.

Benefits of the invention

The benefits of using a crHIV strategy for the genetic therapeutic treatment of AIDS and other diseases are considerable. For example, the problem of targeting the vector to HIV-infected cells is addressed. After in vivo transfection of crHIV into infected CD4 + cells, crHIV are packaged into progeny virions by endogenous infectious HIV envelope proteins. The crHIV RNA binds along the internal progeny virions and infects cell types that are normally infectable by this particular strain of HIV, thus producing non-pathogenic virions. This includes difficult-to-target cells, such as brain microglial cells, which are a major reservoir of HIV infection for the central nervous system. It is likely that crHIV vectors that infect uninfected CD4 + cells have little toxicity because crHIV vectors do not encode any viral proteins. In addition, competition of the crHIV vector with wild-type HIV results in the production of non-pathogenic particles, resulting in reduced viral load. Reducing pathogenic HIV-1 loads not only increases the survival time of infected individuals, but also reduces the rate of transmission to uninfected individuals, since crHIV particles can also expand systemically (ie, like infectious HIV). Reduced pathogenic HIV-1 loads in the blood may be particularly important in pregnant HIV-infected individuals, as crHIV production may also reduce HIV-1 transmission in infected mothers to the fetus in the uterus.

The plasmid / lipid mixture that can be used to introduce the crHIV vector into host cells should be stable and inexpensive to manufacture, avoiding costly ex vivo strategies. Of course, the method of the invention is inherently adaptable for use in the delivery of genes ex vivo, if desired. Regardless, the availability of liposomes opens up the possibility of treating the general population, something that is not feasible with current gene therapy strategies. The crHIV vectors can also be constructed to contain several ribozymes that can be constructed against various targets in the HIV genome. This reduces the possibility that infectious HIV will mutate and thereby escape the effect of anti-HIV ribozymes. In addition, a vector capable of conditionally replicating can be applied to the treatment of other viral infections, particularly those with high virus turnover.

A particularly useful feature of crHIV vectors is that they can be used for post-transcriptional expression of genetic antiviral agents, for example ribozymes. Infection of uninfected cells with crHIV vectors results in lower toxicity, since in the absence of Tat protein there will be little expression from the HIV long terminal repeat (LTR) promoter. High levels of crHIV expression and subsequent antiviral activity only occur when Tat protein is provided by complementation with wild type HIV. Therefore, crHIV vectors are not designed to protect cells from HIV infection, but to reduce the overall burden of wild-type HIV by selective accumulation of non-pathogenic crHIV particles.

While not wishing to be bound by any particular theory relating to the processes and functions of this invention, it is believed that ribozymes can be used, as confirmed in the following examples, to obtain crHIV genomes with a selective advantage, based on two useful properties: (1) have a high degree of specificity, and (2) have a relative potency, depending on their ability to be co-located with target RNAs (Cech, Science, 236, 1532-1539 (1987)). Ribozymes are relatively efficient because they cleave target RNA with high efficiency only when effectively co-located with target RNAs.In mixed HIV / crHIV infection, co-location of ribozymes containing crHIV RNA with wild type HIV RNA because of the HIV RNA genomes before packaging Cleavage of non-genomic wild-type RNAs, which is required for viral protein production, is probably less efficient than cleavage of wild-type genomic RNAs because non-genomic HIV RNAs do not dimerize. In the experiments described herein, it has been discovered that the selective advantage that crHIV acquires is due to the selective packaging of crHIV into viral particles. These results indicate that the most efficient cleavage occurs intracellularly during dimerization, resulting in selective destruction of wild-type HIV RNA by host nucleases. As a result, advantageous packaging of crHIV into viral particles is made possible.

Application of crHIV vectors for HIV therapy may include not only genomic selection of crHIV but also cell selection of cells producing crHIV particles. Otherwise, the cells will be · »·· ·· ΦΦΦΦ

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9 φ · · ♦ ♦ ♦ 9 9 9 9 9 9 9 9 9 9 9 9 9 · · · · · · · · · · · · · · · · · · · · · · · · · , and they will quickly prevail. A selective advantage can be provided by crHIV expressing cells by inserting a gene into crHIV genomes (eg, a multispecific drug resistance gene) that confers crHIV expressing cells (in the presence of a drug) an advantage of survival over cells expressing wild-type HIV. Under these conditions, wild-type HIV-expressing cells progressively die but still produce some amount of wild-type HIV, while crHIV-expressing cells selectively producing crHIV survive. Infection of crHIV containing cells with the remaining wild-type HIV cells will result in further production of crHIV-containing viral particles. Thus, viral genomic shift may result in the cumulative infection of CD4 + cells with the crHIV genome, thereby altering the viral equilibrium in the host cell from pathogenic wild-type HIV to non-pathogenic crHIV genomes. This strategy will result in the disappearance of wild-type HIV once there is a selective shift in the balance of HIV genomes from wild-type HIV to crHIV. Ultimately, viral replication will be discontinued because crHIV can only replicate in the presence of wild-type helper genomes. Thus, under such mutually restrictive conditions, it may be possible to construct crHIV vectors that not only reduce the viral load of wild-type HIV, but also remove the virus from the HIV-infected host.

Means of production

The vector may be produced by a complementation method, either using transient transfection of the vector and helper genome into a cell line, preferably but not limited to a known 293T cell line, or using a enveloping cell line. The cell is transiently transfected with high transfection efficiency with a transfection agent, preferably calcium phosphate, electroporation, or a lipid transfection agent. When transfection occurs, the vector is isolated from the supernatant preferably no earlier than 12 hours after transfection and no longer than 7 days after transfection. The vector is, if desired, concentrated by high speed centrifugation without precipitation or ultracentrifugation. Preferred centrifugation conditions are about 5000 to 12,000 x g, preferably about 10,000 x g. More preferred conditions are overnight centrifugation at 4 ° C.

44 ··

4 4

4 44 4

4444 ► 4 4

I 4 4

Methods of vector purification

Method 1

1. Clarification of the viral supernatant

2. Concentration by ultrafiltration

3. Diafiltration with or without benzonase treatment

4. Ion exchange chromatography

5. Size exclusion chromatography or diaultrafiltration

6. Possible concentration by ultrafiltration

Method 2

1. Clarification of the viral supernatant

2. Ion exchange chromatography

3. Diafiltration or size exclusion chromatography

4. Possible benzonase treatment

5. Size exclusion chromatography or diafiltration

6. concentration by ultrafiltration, if applicable

Various resins can be used for the above procedures, including Poros 50HQ from Perseptive Biosystems. Hollow fiber cartridges, including cartridges (UFP-750 series) from A / G Technologies, can be used for ultrafiltration or diafiltration.

Routes of administration

According to the invention, when gene therapy for viral infection is desired, the vector is introduced into a host cell as previously described. The method of introduction comprises contacting a host cell capable of being infected with a virus with a vector of the invention. It is preferred that the contact includes any method by which the vector is introduced into the cell; the method is not dependent on the particular deployment means and cannot be interpreted. Methods of introduction are known to those skilled in the art and are also exemplified herein.

Accordingly, the introduction can be performed, for example, either in vitro (eg, in an ex vivo gene therapy method) or in vivo, including the use of electroporation, transformation, transduction, conjugation or three-parent pairing, transfection, infection, • 444

4444 »1» 444 • * 4 9 4 44 9 • 4 4444 «4« 4 * 4444 4444 4 4444 4 4 4444 4 · 44 «94« 4 44 membrane fusions with cationic lipids, high-speed bombardment projectiles, coated with DNA, incubation with DNA, precipitated calcium phosphate, direct microinjection into single cells, etc. Other methods are available and known to those skilled in the art.

Preferably, however, the vectors or ribozymes are introduced by cationic lipids, e.g., liposomes. Such liposomes are commercially available (eg, Lipofectin ™., Lipofectamine ™, and the like, supplied by Life Technologies, Gibco BRL, Gaithersburg, Md.). In addition, liposomes having increased transfer capacity and / or reduced toxicity in vivo (e.g., as described in PCT Patent Application No. WO 95/21259) may be used in the present invention. For the administration of liposomes, the recommendations described in PCT Patent Application No. WO 93/23569 may be used. Generally, with such administration, the formulation is taken by most lymphocytes within 8 hours at 37 ° C, with more than 50% of the injection dose detected in the spleen one hour after intravenous administration. Similarly, other pharmaceutical carriers include hydrogels and controlled release polymers.

The form of the vector introduced into the host cell may vary, depending in part on whether the vector is introduced in vivo or in vitro. For example, the nucleic acid may be a closed ring, interrupted or isolated, depending on whether the vector is to be maintained extragenomically (i.e., as an autonomously replicating vector), integrated as a provirus or prophage, transiently transfected, transiently infected using a replication deficient or conditionally of a replicating virus, or stably introduced into the host genome via a double or single recombination cross.

Prior to introduction into the host, the vector of the invention may be formulated in various compositions for use in both therapeutic and prophylactic treatment methods. In particular, the vector can be incorporated into pharmaceutical compositions by association with suitable, pharmaceutically acceptable carriers or fillers and can be formulated to be suitable for human or veterinary application.

Thus, the composition for use in the method of the invention may comprise one or more of the aforementioned vectors, preferably in association with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are known to those skilled in the art, as are suitable routes of administration. The choice of carrier can be determined in part by the particular vector, as well as by the particular method used to administer the composition. One skilled in the art will recognize that there are different routes of administration of the composition, and although more than one route of administration may be used for administration, a particular route of administration may provide a more immediate and effective response than another route. Accordingly, there are many suitable dosage forms for the compositions of the present invention.

• w ·· + «·· ·»

9

9999 «8 ·

9 9 9 9 9 • 9 9 9 9 9

The composition comprising the vector of the present invention, alone or in conjunction with other antiviral compounds, may be formulated in a form suitable for parenteral administration, preferably intraperitoneal administration. The dosage form may include both aqueous and non-aqueous forms, isotonic sterile injectable solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the dosage form isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solvents, thickeners, stabilizers and preservatives. These dosage forms may be presented in unit doses or in multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (freeze-dried) state requiring only the addition of a sterile liquid carrier, for example water for injection, immediately prior to use. Ready-to-use injectable solutions and suspensions may be prepared from sterile powders, granules and tablets as described herein.

The vector may be stored in any suitable solution, buffer, or lyophilized form, if desired. A preferred storage buffer is Dulbecco's Phosphate Buffered Saline; Dulbecco's Phosphate Bufered Saline mixed with a 1 to 50% trehalose in water (1: 1) solution, preferably a 10% trehalose in water (1: 1) solution, so that the final concentration is 5% trehalose; Dulbecco's Phosphate Buffered Saline mixed with a 1 to 50% glucose in water (1: 1) solution, preferably a 10% glucose in water (1: 1) solution, such that the final glucose concentration is 5%; 20mM HEPES buffered saline mixed with 1 to 50% trehalose in water (1: 1), preferably 10% trehalose in water (1: 1) so that the final trehalose concentration is 5% or Dulbecco's Phosphate Buffered Saline mixed with 1 to 1 A 50% solution of mannitol in water (1: 1), preferably a 5% solution of mannitol in water (1: 1) such that the final concentration of mannitol is 2.5%.

The dosage form suitable for oral administration may consist of liquid solutions, such as an effective amount of a compound dissolved in a solvent such as water, saline or fruit juice; capsules, sachets or tablets containing a prescribed amount of the active ingredient, in solid form or granules; solutions or suspensions in an aqueous liquid; and oil-in-water or water-in-oil emulsions. Tablet forms may contain one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silica, talc, magnesium stearate, stearic acid and other fillers, coloring agents, diluents, buffering agents, wetting agents agents, preservatives, flavoring agents, and pharmaceutically acceptable carriers.

Aerosol dosage forms may also be prepared for administration by inhalation. The aerosol dosage form may be placed in a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen and the like.

to •• this this this ··· * totototo • to the to to this to this to this to this to ·············· ·· ··

Similarly, oral dosage forms may include curd forms, which may contain the active ingredient in the flavoring component, usually sucrose and acacia and tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia and mouthwashes containing the active ingredient in a suitable liquid carrier, as well as creams, emulsions, gels and the like, containing, in addition to the active ingredient carriers known in the art.

Topical dosage forms may be in the form of creams, lotions or lotions.

Dosage forms for rectal administration may be in the form of suppositories with a suitable base comprising, for example, cocoa butter or salicylate. Dosage forms suitable for vaginal administration may be pessaries, tampons, cream, gel, paste, foam or spray dosage forms containing, in addition to the active ingredient, carriers known in the art to be appropriate. Similarly, the active ingredient may be combined with a lubricant such as a condom coating.

The dose administered to an animal, especially a human, according to the present invention should be sufficient to elicit a therapeutic response in the infected individual for a reasonable period of time. The dose is determined by the strength of the respective vector used for treatment, the extent of the disease state, as well as the body weight and age of the infected individual. Dose size will also be determined based on the existence of any adverse side effects that may accompany the use of a particular vector. It is always desirable to keep the adverse side effects to a minimum whenever possible.

The dosage may be in unit dosage form, such as tablets or capsules. The term "unit dosage form" is used herein to refer to physically discrete doses suitable as unitary unitary doses for both human and animal subjects, each unit containing a prescribed amount of the vector, alone or in conjunction with other antiviral agents, in an amount calculated as sufficient to induce the desired effect in association with a pharmaceutically acceptable filler, carrier or composition. The specification of the unit dosage forms of the present invention depends upon the particular compound or compounds being employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in a given host. The dose administered should be in an "antiviral effective amount" or in an amount necessary to achieve an "effective level" in an individual patient.

Although the "effective level" is used as the preferred endpoint for dosing, the actual dose and dosage regimen may vary, depending on differences in pharmacokinetics between individual patients, drug distribution and metabolism. "Effective level" can be φφφφφφ «• φφφφφφφ

ΦΦΦ ΦΦΦ • • • •

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For example, defined as the blood or tissue level required in a patient that corresponds to the concentration of one or more vectors of the invention that inhibit a virus, such as HIV, in a test for the predictive clinical antiviral activity of these chemical compounds. The "effective level" for the compounds of the present invention may also change when the compounds of the present invention are used in conjunction with zidovudine or other known antiviral compounds or combinations thereof.

One of skill in the art can readily determine the dosage, dosage regimen, and route of administration for a particular dosage form of the compound being used to achieve the desired "effective level" in an individual patient. One of ordinary skill in the art will also readily determine and use an appropriate indication of the "effective level" of the compounds of the present invention, either directly (e.g., by chemical analysis) or indirect (e.g., surrogate indication of viral infection such as p24 or reverse transcriptase for AIDS or related diseases). AIDS) by analyzing appropriate samples taken from the patient (e.g., blood and / or tissue).

In addition, in view of determining an effective level in a patient for the treatment of AIDS or AIDS-like diseases, animal models have been widely available that have been extensively incorporated into the in vivo efficacy evaluation against HIV in various gene therapy procedures (Sarver et al. above). These models include mouse, donkey and cat. Although these animal models are not naturally susceptible to HIV disease, chimeric mouse models (eg, SCID, bg / nu / xid, NOD / SCID, SCID-hu, immunocompetent SCID-hu, BALB / en bone marrow harvested) reconstituted with human peripheral blood mononuclear cells (PBMCs), lymph nodes, fetal liver / thymus, or other tissues can be infected with vector or HIV and used as models for HIV pathogenesis and gene therapy. Similarly, the simian imudodeficiency virus (SIV) or donkey model can be used, as well as the fox or cat immunodeficiency virus model.

These models can also be used to determine vector safety for vector system evaluation procedures in clinical procedures. An important application is the use of these animal models for biodistribution studies. Transduced cells, preferably but not limited to human cells containing the vector, are injected into a non-human animal model, and the safety of the vector is determined by the absence of vector genetic material in animal tissue. This absence of vector genetic material in animal tissue means that this vector does not autonomously replicate in the absence of helper or wild-type virus and can therefore be considered safe for clinical use in humans. The presence of a vector in the absence of a helper vector or helper virus can be considered a security risk unless the vector is not expected to replicate autonomously. However, when vectors are expected to be capable of autonomous replication, other safety criteria, such as inability to replicate in certain tissues or level of replication in an animal, need to be determined. The presence or absence of this vector can be determined by PCR or FACS analysis if the test vector expresses a marker gene that can be visualized by FACS but is not limited to these detection methods.

Generally, the amount of vector suitable to achieve a concentration of the administered ribozyme (or vector) in the tissue is preferably from about 5 pg / kg to about 300 mg / kg body weight per day, particularly from about 10 pg / kg to about 200 mg / kg body weight. weight per day. In certain applications, e.g. topical, ocular, or vaginal, multiple daily doses are preferred. In addition, the number of doses will depend on the mode of delivery and the particular vector administered.

In the treatment of some virus-infected individuals, it may be desirable to use a "mega-dose" regimen where a large dose of the vector is administered, allowing the compound time for action and then administering the appropriate agent to inactivate the active compound (s). In the method of the invention (i.e., replication of the vector in competition with the virus being treated), this treatment is necessarily limited. In other words, as the level, such as HIV, decreases, the level of HIV-dependent vector for virion production will also decrease.

The pharmaceutical composition may contain, in conjunction with the vector of the invention, other pharmaceutical compounds when used for the therapeutic treatment of AIDS. These other pharmaceutical compounds can be used in their traditional manner (ie as agents for the treatment of HIV infection), as well as in particular for selection for crHIV viruses in vivo. This selection described herein will promote the proliferation of conditionally replicating HIV and allow the conditionally replicating HIV to compete more effectively with wild type HIV, which will necessarily limit the pathogenicity of wild type HIV. In particular, it is contemplated that an antiretroviral agent such as preferably zidovudine may be employed. Other representative examples of these additional pharmaceutical compounds that may be used in addition to those described above include antiviral compounds, immunomodulators, immunostimulants, antibiotics and other agents and treatment regimens (including those referred to as alternative medicine) that can be used to treat AIDS . Antiviral compounds include, but are not limited to, ddl, ddC, ganciclovir, fluorinated dideoxynucleotides, non-nucleoside analogue compounds such as nevirapine (Shih et al., PNAS, 88, 9878-9882 (1991)), TIBO derivatives such as R82 913 (White et al., Antiviral Research, 16,257-266 (1991)) and BI-RJ-70 (Shih et al., Am. J. Med., 90 (Suppl. 4A), 8S-17S (1991)). Immunomodulators and immunostimulants include, but are not limited to, various interleukins, CD4, cytokines, antibody preparations, blood transfusions, and cell transfusions.

·· ···· · · · · · ·

Antibiotics include, but are not limited to, anti-fungal agents, antibacterial agents, and anti-Pneumocystis carinii agents.

Administration of a virus inhibiting compound with other antiretroviral agents, and in particular with known RT inhibitors, such as ddC, zidovudine, ddl, ddA or other inhibitors that act against other HIV proteins, such as anti-TAT agents, will generally inhibit most or all replication stages of the virus life cycle. DdC and zidovudine dosages used in AIDS or ARC patients have been reported. The virostatic range ddC is generally between 0.05 μΜ and 1.0 μΜ. The range from 0.005 to 0.25 mg / kg body weight is virostatic in most patients. Dosage ranges for oral administration are somewhat broader, for example 0.001 to 0.25 mg / kg administered in one or more doses at intervals of 2.4, 6, 8 and 12, etc. hours. It is preferred that 0.01 mg / kg body weight of the patient be administered ddC every 8 hours. For combination therapy, the additional antiviral compound may, for example, be administered concurrently with the vector of the invention or the dosage may be adjusted as desired. The vector may also be combined in a composition. If a combination is used, the dose of each component may be lower than when taken alone.

Overview of stamps in the drawings

Figures IA to 1K are schematic representations of particular improved conditionally replicating vectors included in the invention: pN1 (cPT), pN1 (cPTc) ASenvGFP (464), pN1 (cPT) ASenvGFP (452), pN1 (cPT2) ASenvGFP, pN1 (cPT) cGFP, pN1 GFP (cPT) T, pN1 (cPT) GFPTAR, pN1 GFP (cPT) VT, pN2GFP, pN2ASenvGFP (418) and pN2 (spe) ASenvGFP. Of course, the green fluorescent protein (GFP) marker gene can be removed before using the vector in the applications described herein. Indication: N1, a minimal HIV-derived vector without gag / pol sequences but with a gag coat sequence indicates that gag 'or gag and the stop codon are located about 40 pairs of bases from the AGT gag sequence; N2, an HIV-1 derived vector, capable of expressing gag / pol sequences; AS, antisense; Asenv, env sequence present in antisense orientation; gag, pol and env, sequences encoding proteins that make up the virus envelope, reverse transcriptase, and envelope, respectively; tat, rev, rre and nef, other viral genes; cPTc, minimal intermediate polypyrimidine stretch of sequence; cPT, a central polypyrimidine stretch containing a large 548 base pair insert; cPT2, a central polypyrimidine region containing a 438 base pair sequence (does not contain gag sequences such as cPT); the spe, gag / pol sequences are not translated; GFP, encoding a green fluorescent protein.

• • •

Figure 2 describes DNA sequences of the wild-type HIV U5 RNA of SEQ ID NO: 1 (A) and the modified crHIV U5 RNA of SEQ ID NO: 2 (B). The numbers indicate the number of runs since the start of transcription.

Figure 3A to 3E depicts the effects of a helper vector on a conditionally replicating vector (cr) in relation to the titer of the cr vector produced. Figure 3 A shows a molar ratio of 1: 0.5 with the highest titer result for pN1 (cPTc) GFP; 3B and 3C show a molar ratio of 1: 0.75, providing the highest titer for pN1 (cPT) GFP and pN1 (cPT2), respectively. ASenvGFP and 3D and 3E show a molar ratio of 1: 1.5 giving the highest titers of pN1cGFP and respectively. pN2cGFP. These vectors are shown in Figure 1, with the exception of pN1 (cPT) GFP lacking the antisense env sequence and pN1cGFP and pN2cGFP having the inserted cytomegalovirus (CMV) promoter for GFP expression. These figures indicate that with a biplasmid system, conditions for producing titers of at least 1.5 x 10 transduction units per milliliter were achieved.

Figures 4A and 4B show maps of two HIV-2 based conditionally replicating vectors: pSIcGFP and pS2cGFP. PSI and pS2 designate the presence or absence of gag / pol sequences as described above for pN1 and pN2. The designation c indicates the presence of a CMV promoter to direct GFP expression.

Figure 5A and 5B depict the effects of various helper vector constructs on packaging of pSIcGFP and pS2cGFP. The effect of different molar ratios between the helper vector and the conditionally replicating vector was tested together with the effect of different RREs on the helper vector. As noted, the use of a 1: 1 ratio of the helper vector to the conditionally replicating vector was more effective in producing functional vector particles than the other ratios used.

Figures 6A to 6G depict structures of various helper vector constructs: pVIRPAC-1, pVIRPAC-2, pVIRPAC-1.1Rz, pVIRPAC-1.2, VirPac1.2Rz, pVIRPAC-1.2Rz2, and pVIRPAC-1.2RzIn, as included in the present invention . Rz denotes the presence of anti-U5 ribozymes, while 1,1 and 1,2 denote a helper vector having RRE derived from HIV-1 and HIV-2, respectively.

Figure 7 shows the effect of one or more ribozymes on viral vector titers. PN1 (cPT) GFP was enveloped in HeLa-tat cells in the presence of pVirPac1.2 (containing no ribozymes), pVirPac1.2RzIn (containing one ribozyme and intron), pVP1.2Rz (containing one ribozyme) or pVP1.2Rz2 (containing two ribozymes) ). As shown in the graph, the presence of a single ribozyme (pVirPac1.2Rz) or ribozymes and an intron designed to affect helper RNA cellular transport (pVirPac1.2RzIn) had no conclusive effect on the vector titer. PCR analyzes of the titer of the samples of the co-packaged helper vector constructs indicated low co-packaging in the helper vector. the presence of ribozymes against high spoiling in the absence of ribozymes. The “VirPac” indicator can also be referred to as “VIRPAC” or “VP”.

Figure 8 shows the inhibitory effect of vectors from pN1 and pN2 series on wild-type replication in T cells. T cells were first transduced with the vector and then infected with wild-type virus at a multiplicity of infection of 0.1 with 100% transduced cells. Virus replication was tested using p24 ELIS A antigen capture analysis. pN2ASenvGFP showed profound inhibition of wild-type virus replication.

Figures 9A and 9B show robust inhibition of wild-type HIV replication with pN1 and pN2 based vectors. Figure 9A shows robust inhibition of wild-type HIV in human T cells by the pN1GFP (cPT) VT and pN2ASEnvGFP vectors as compared to control tumor cells after infection with wild-type HIV. Figure 9A shows similar results in T cells with pN1 (cPT) ASenvGFP and pN2ASenvGFP.

Figure 10A and 10B show vectors and their use for selecting transduced cells. Figure 10A shows the organization of the vectors used where pNICMIG and pNIMCG contain an internal CMV promoter, while pNIMIG and pNIMIG-W express MGMT genes via the HIV LTR promoter. Figure 10B shows a graph of expansion of SupT1 cells transduced with the above vectors and selected on the basis of BG and BCNU as described in Example 5 below.

Figure 11, panels A to F, show selection of transduced primary CD4 + cells by BG and BCNU. CD4 + cells transduced with pN2MIG were cultured at 0; 0.5; 2.5; 10 and 10 pM BG (shown on panels A to F), with the indicated BCNU concentration. Panel F further shows the results of a 1: 5 dilution of transduced cells followed by treatment with 10 μΜ BG at the indicated BCNU concentrations. The baseline level of 3% GFP + cells in panel F was increased at least 32-fold to a 97% level that has not yet been observed in vitro.

Figure 12 shows the effects of ribozymes in protecting against co-packaging of helper RNA in vector preparations. Virus-containing supernatants were extracted by boom extraction and subjected to DNase I digestion, followed by qualitative reverse transcriptase-PCR (RT PCR) detection of the HIV-1 gag-pol region found in the helper construct. As can be seen, the presence of one or two ribozymes in the helper construct used (pVP1.2Rz or pVP1.2Rz2) significantly reduced the amount of co-packaged helper vector relative to the helper-free ribozyme (pVP1.2).

Figures 13A and 13B show the ability to remove tumor cells from CD34 + stem cells. Figure 13A shows that at an MOI of 10, pN1 (cPT) ASenvGFP transduced 98.52% of SupT1 tumor cells after a single cycle of transduction. Figure 13B shows that significant transduction was not evident in CD34 + cell transduction after a single transduction cycle. Significant transduction was observed after three cycles of transduction. Labeling: for example, 1/2 / A. indicates 1 cycle of transduction, an MOI of 2 and the presence of viral complement proteins on the transduced vector, while for example 3/50 means 3 cycles of transduction and an MOI of 50.

Figure 14A shows the structures of wild-type VSV-G, wild-type RDI 14 and chimeric envelope proteins with designated extracellular, transmembrane and cytoplasmic domains. Figure 14B shows titers of HIV-1 vectors pseudotyped in HT1080 with various envelope proteins (VSV-G wild type, rabies G virus, wild type RD 114a RD114E, chimeric VSV-G and RD114 constructs).

Figures 15A to 15E are schematic representations of specific envelope line constructs included in the invention: p (CGCRSRRE), p (TREtTApuro), p (BI-RevTat), p (EH-GP) and p (CMV-GP). Figure 15F shows the organization of rev-dependent VSV-G constructs.

Figure 16 shows the yield of pN1 (cPT) GFP vectors on represented cells before and after concentration in HeLa-tat cells. As can be seen, the yield is maintained at about 10 ¹⁰ for each set of conditions.

Figure 17 shows the results using the Rev / RRE / CRS system to control VSV-G expression.

Figure 18 shows the effect of stock buffer on vector yield when stored for 3-5 weeks at different temperatures. As can be seen, the presence of 10% trehalose or 10% glucose in either D-PBS or HBS gave good vector yields when stored at either -20 ° C or -80 ° C.

DETAILED DESCRIPTION OF THE INVENTION

The present inventive compounds and methods are further described in connection with the following examples. These examples serve to further illustrate the present invention and are not intended to limit the scope of the invention.

Example 1

This example describes the construction of conditionally replication competent vectors of the invention. Specifically, this example describes the construction of conditionally replicating HIV-based vectors, i.e., crHIV vectors.

One of the most striking aspects of HIV-1 pathogenesis is the production of genetic variants of the virus. The rapid production of HIV variants in vivo suggests that this virus can be viewed as “9” 999 • · · ·

9,999 in Darwin's genetic modeling (see, eg, Coffin, Curr. Top. Microbiol. Immunol., 176, 143-164 (1992) and Coffin, Science, 267, 483-489 (1995)). These variants are the result of inaccuracies of the HIV-1 reverse transcriptase molecule, which generates mutations in provers newly transcribed from viral genomic RNA. Therefore, under in vivo conditions, characterized by the absence of a significant critical period and many replication cycles, there is a substantial degree of genetic variability with the production of many viral variants. Nevertheless, the wild type of HIV prevails, because under such unlimited conditions it has the highest selective advantage. However, in the presence of an inhibitor, for example zidovudine, a viral variant will be selected which is provided with a higher selective advantage than the wild-type strain and as a result will prevail (Coffin (1992) and (1995) supra). On this basis, the present invention provides a conditionally replicating vector strategy that offers non-pathogenic HIV-1 genomes with a selective advantage over pathogenic wild-type HIV-1.

These non-pathogenic, conditionally replicating HIV (crHIV) vectors are defective HIV that are subject to replication and envelope only in cells that are infected with wild-type HIV. These crHIV genomes compete with wild type HIV and reduce its viral load. This effect of reducing viral load of wild-type HIV in an infected host should lead to an increase in life expectancy. It should also reduce the ability of the infected host to transmit wild type HIV to uninfected individuals. Two factors seem important for successful competition of crHIV with wild-type HIV-1: (1) the selective advantage of crHIV genomes over wild type HIV genomes, and (2) the selective advantage of crHIV expressing cells over wild-type HIV expressing cells (i.e. a selective advantage for the production of crHIV virions by crHIV expressing cells over cells expressing wild-type HIV).

These crHIV vectors are conditionally replicated due to the fact that they contain sequences required for RNA expression, dimerization and packaging but do not express functional (i.e., wild-type) HIV-1 proteins. A selective advantage has been incorporated into these crHIV vectors by inserting a ribozyme cassette that cleaves the U5 region of the wild-type HIV genome but not the U5 region in the crHIV RNA.

The ribozymes present in the vectors do not cleave the crHIV RNA because the U5 region of the crHIV RNA has been modified by the substitution of conserved bases (substitution of bases present in other HIV strains) to prevent ribozymes from effectively binding and cleaving these sites. Moreover, these crHIVs are non-pathogenic because they do not encode proteins believed to be responsible for CD4 + cell death. When HIV-infected cells (which have been transfected with the crHIV vector) are activated, they become capable of complementing crHIV genomic deficits, resulting in the production of viral offspring of crHIV.

• ··· ·· · to · to · ···

CrHIV genomes were generally constructed over the entire length of the infectious HIV clone pNL4-3 (Adachi et al. (1986), supra). All cloning reactions and DNA, RNA and protein manipulations were performed using methods known to those skilled in the art, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory, NY (1982)) . The enzymes and reagents used in these reactions were obtained from commercial suppliers (eg, New England Biolabs, Inc., Beverly, Mass .; Clontech, Palo Alto, Calif., And Boehringer Mannhiein, Inc., Indianopolis, Ind.) And have been used according to manufacturers' recommendations. In addition, the maintenance and propagation of the vectors was carried out by methods that are generally known and previously described (eg, Dropulič et al. (1992), supra, and Dropulič et al. (1993), supra).

pNL4-3 was digested with Pst I (which cleaves at gag, at about +1000 from the transcription start) and Xho I (which cleaves at nef, at about +8400 from the start of transcription) and a polylinker containing appropriate restriction sites was inserted . A 0.86 kb Bgl II to Bam HI fragment containing a reversible element (RRE) was cloned into the Bam HI site present in the polylinker. These manipulations resulted in the deletion of the wild-type HIV genome from sites in the gag coding region to sites in the U3 coding region (i.e. also a deletion of the nef gene). While this vector is capable of producing a truncated gag transcript, the full length of the functional Gag protein is not produced by this vector. However, since the wild-type Gag functions of the invention are not necessary, gag sequences may be mutated to prevent translation of the Gag protein.

A ribozyme cassette as described herein containing either one or more ribozymes was inserted into the Sal I site downstream of the Bam HI site. To do this, ribozyme sequences encoding complementary deoxyoligonucleotides were synthesized, linked, and then cloned into a Sal I site. The ribozymes used to construct crHIV vectors were hammerhead ribozymes. These ribozymes contained a catalytic domain composed of 22 base pairs and two hybridization domains containing 9 base pairs each. Ribozymes were targeted to either +115 or +133 (i.e., corresponding to the number of base pairs downstream of the start of transcription) of the U5 RNA sequence. The hybridization domains and the catalytic domain (underlined) of ribozymes directed against the +115 site and +133 site are as follows:

CACACACAACACTGATGAGGCCGAAAGGCCGAAACGGGCACA ("+115 ribozyme") SEQ ID NO: 3

ATCTCTAGTCTGATGAGGCCGAAAGGCCGAAACCAGAGTC ("+133 ribozyme") SEQ ID NO: 4

The ribozyme cassette contained either single, double or triple ribozyme (s), arranged in tandem. Vectors containing either single or triple ribozymes can be readily constructed and targeted to the same U5 HIV RNA site, at position +115. Vectors containing double ribozymes can be readily engineered and targeted either to the same site at +115 or to different sites at +115 and +133 U5 of HIV RNA.

In order to complete the construction of these vectors, crHIV vectors were given resistance to cleavage of ribozymes (i.e., if in the form of RNA) by mutating a site recognized by the hammerhead ribozymes occurring in the U5 region of the crHIV genome. To achieve this, a double-stranded oligonucleotide (i.e.

AAGCTTGCCTTGCGTGCTCAAAGTAGTGTGTGCCCACCTGTTGTGTGACTCTGGCAG CTAGAGATCCCACAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCC (SEQ ID NO: 13), comprising the base substitutions shown in Figure 2, shown in Figure 2. Specifically, substitutions were made at the hybridization and ribozyme cleavage sites at base pairs 115 and 133. In particular, as shown in Figure 2, mutations were made at base pairs 113,114,132, 134 and 142. These sites may be modified to contain any mutation (i.e. GTGTGCCCNNCTGTTGTGACTCTGGNANCTAGAGANC, where N can be any mutant nucleotide, SEQ ID NO: 14). However, it is preferred that this sequence be mutated such that, for example, G is substituted by A at position +113 (i.e., such sequence comprises GTGTGCCCATCTGTTGTGACTCTGGTAACTAGAGATC, SEQ ID NO: 15), substitution U (i.e. T for DNA sequence) after C at site +114 of SEQ ID NO: 5, substitution of U (i.e., T for DNA sequence) after C at site +132 of SEQ ID NO: 6, substitution of A on G at +134 (i.e. it comprises GTGTGCCCGTCTGTTGTGACTCTGGTAGCTAGAGATC, SEQ ID NO: 16) and a U (ie T for DNA) substitution for A at +142, which mutations can be made either alone or in combination. In particular, in the absence of other U5 mutations, the U substitution (i.e., T for DNA sequence), at +114 to C at +114 of SEQ ID NO: 5 and / or at +132 of SEQ ID NO: 6 in crHIV US RNA prevents its cleavage by ribozymes (Uhlenbeck (1987), supra). Inverted base substitutions are present in various other strains of HIV (Myers et al., HIV Sequence Database, Los Alamos Nat. Lab. (1994)), suggesting that these substitutions do not reduce the replication capacity of the HIV genome.

The method disclosed herein can be used to construct other conditionally replicating vectors, for example, containing different viral genomes (eg, different RNA viruses) or different genetic antiviral agents. In addition, the conditionally replicating vector may further be »4441

4

444 modified to give the host cell into which the vector is incorporated a selective advantage over the host cell containing the wild-type virus. The vector may, for example, be modified to also encode multispectral resistance to drugs or to a mutated protease or reverse transcriptase.

Of course, these methods can also be used to construct the various helper vector contracts described herein.

Example 2

This example describes resistance to ribozyme cleavage of conditionally replicating vectors, particularly crHIV vectors.

In vitro transcription was performed to confirm the resistance of crHIV vectors to ribozyme cleavage. To perform this, ribozyme sequences were cloned at the Xho I site of pBluescript KSII (Stratagene, La Jolla, Calif.). A 0.21 kilobase pair (kb) Bgl II fragment containing the mutated crHIV U5 region was similarly excised from the crHIV vector and inserted into the Bam HI site of pBluescript KSII. The resulting modified pBluescript KS II vectors were linearized with Bss HII prior to in vitro transcription. A similar plasmid expressing wild-type HIV U5 RNA (described in Myers et al. (1994), supra) was used as a control. It was linearized with Eco R1 prior to in vitro transcription.

Radioisotopically labeled U5 HIV RNA and ribozyme RNA were obtained by in vitro transcription of these vectors as described (Dropulié et al. (192), supra). Isotopically labeled transcripts were co-incubated (at a molar ratio of target to ribozyme of 1: 2) in 1X transcription buffer containing 40 mM Tris.HCl, pH 7.5.6 mM MgCl ₂ , 2 mM spermidine, and 10 mM MaCl. The samples were heated to 65 ° C and then cooled to 37 ° C for 5 minutes before adding a stop buffer solution containing 95% formamide, 20 mM EDTA, 0.05% Bromphenol Blue and 0.05 Xylene Cyanol FF. The reaction products were then resolved by polyacrylamide gel electrophoresis (PAGE) in a denaturing medium and detected by autoradiography.

When wild-type U5-HIV RNA was incubated with a transcript containing a single ribozyme at +115, easy cleavage was observed. This cleavage resulted in PI and P2 products. Cleavage can also be observed when wild-type HIV RNA has been incubated with RNA containing double ribozymes either at the same site or at different sites. When a ribozyme containing a transcript directed to two different sites was incubated with wild-type HIV RNA, products P1, P2 and P3 were produced. P3 results from cleavage at +133.

For comparison, when the modified U5 containing crHIV RNA was incubated with either a single ribozyme directed at +115 or a double ribozyme directed

either at +115 or +133, no cleavage products were detected. Thus, these results confirm that crHIV U5 RNAs are resistant to ribozyme cleavage, whereas wild-type HIV-U5 RNA is cleaved by anti-U5 ribozymes. In addition, these results confirm that the approach of the present invention can be used to impart a selective advantage for replication to conditionally replicating vectors (including vectors other than crHIV vectors) when introduced into a host cell as compared to a wild-type virus strain.

Example 3

This example briefly describes the key steps in the production of the helper vector constructs of the invention. Unless otherwise indicated, nucleic acid sequences encoding various retroviral elements were obtained from publicly available sources. All described steps required sequencing of the entire coding regions for all proteins to detect errors and make corrections if necessary.

To degenerate the tat sequence and allow it to be targeted by anti-tat ribozymes, plasmid pTAT was mutated using the QuickChange kit from Stratagene to include target sites in the anti-tat ribozyme cassette. The mutated tat sequence was then cloned in an expression vector based on the pMG plasmid from InvitroGen. The tat sequence was fused to IRES in the first ATG using the NcoI site. The vector was further modified to contain the ampicillin resistance gene and the SV40 origin of replication.

The sequences containing the HIV-1 rev and RRE elements were obtained by cutting out the third exon from the rev together with the RRE from pNL4-3, and then cloned into the pLITMUS38 plasmid. The Rev sequence after nucleotide 208, which contains the second exon and part of the third exn up to the BamHI site, was constructed from 3 synthetic nucleotides by ETR and PCR. The PCR produt was cloned in pUC18 and then subcloned in pLIT / RRErev3. These two parts of the rev were fused using the QuickChange kit. The HIV-2 RRE was substituted for the HIV-1 RRE and both constructs were cloned in the gag / pol / Rz vector. The degenerate rev sequences were also directly used for subcloning! in a packaging construct for vectors such as pVIRPac-2.

To degenerate the first 42 nucleotides of the gags that are required for packaging and must be part of the vector plasmid, the BssHII / SpeI fragment from pNL4-6 was subcloned in pLITMUS38. The gag sequence was degenerated using the QuickChange kit so that all of the envelope signal components were eliminated and the HIV major splice donor was reintroduced before the first gag ATG codon. The degenerate gag was subcloned in the plasmid pNgp, donated by Dr. Conde, at the 5 'LTR position to produce a contiguous gag sequence. This resulting construct still contains the splice acceptor (SA) and splice donor (SD) sequences used to express the vif protein. The result of this splicing may be the presence of a ribozyme cassette in the spliced mRNA, which is undesirable due to the possible splitting of the spliced retroviral (conditionally replicating) vector RNA and the resulting decrease in the enveloped transducing vector titer. Overlapping PCR was used to mutate SA and SD sequences. The PCR product was inserted and cloned in the gag / pol construct, followed by insertion of the anti-U5 ribozyme cassette and rev and RRE elements.

Envelope stomatitis G env protein (VSV-G) was cloned into the first NCS from the pMG-tat vector to create an expression envelope cassette. To simplify further cloning steps, VSV-G was cloned as a blunt-ended fragment into a BglII site filled with Klenow polymerase. This resulted in a construct with SV40 origin of replication (ori). To complete the enveloping plasmids as helper vectors, the rev sequence in the second MCS was subcloned. The resulting plasmid was named pVIRPAC-2 (see Figure 6).

In order to generate helper vectors from some enveloping plasmids, the gag / pol / Rz / RRE / rev cassette was subcloned in pMG-tat-G plasmids (without SV40 ori). RRE constructs were prepared from either HIV-1 or HIV-2 (pVIRPac-1.1 and 1.2Rz in Figure 6). As a control for ribozyme function testing, the ribozyme cassette in the final product containing HIV-2 RRE was deleted to give pVIRPac-1,2. Finally, to test the hypothesis that the placement of gag / pol RNA in the splicing mechanism may prevent cleavage of the retroviral (conditionally replicating) vector genomic RNA, the β-globin intron was inserted before the SV40 poles A signal (pVIRPac-1,2RzIn). In other embodiments of the invention, of course, the inserted intron is derived from HIV, preferably from a complete HIV intron that has been confirmed to direct RNA to a cellular splicing mechanism (eg, a spliceosome).

Example 4

This example describes the efficacy of various helper vector constructs. Standardized procedures for transient transfection production of the retroviral vector were used in these experiments. Plasmid DNA megapreparations were prepared using known methods. For virus production, 293T cells were transfected together with the plasmids of the retroviral vector and the helper vector using the calcium phosphate method. Cell supernatants were pooled approximately 40 h after transfection, filtered through 0.45 µm filters in syringes and titrated on HT1080 cells. The vector titer was calculated based on its transduction efficiency, measured by flow cytometry 72 h after infection. To calculate the titer, • * · 4 »· ·

I * * 9 used a formula N * 400000 *, where N is the proportion of transduced cells, 400000 is the approximate number of cells at the time of infection and F is the dilution factor,

To enhance the safety of the helper vector by reducing the possibility of RCV formation upon recombination with the HIV-1-derived retroviral vector, an anti-U5 ribozyme cassette was inserted into certain helper vector constructs. This ribozyme can cut and destroy the retroviral RNA vector when packaged together in virions. However, it is also possible that this ribozyme can also cleave vector RNA within production cells, thereby interfering with virus production. To further enhance safety, the RRE-2 helper constructs included an element to further reduce the likelihood of co-packaging of the helper and vector into viral particles. These helpers were used to package the pN1 (cPT) GFP vector.

As shown in Figure 7, the presence of ribozyme on the helper construct had little effect on the vector titer. Importantly, a helper construct (pVirPac1, 2RzIn) containing an intron to affect cellular transport of helper RNA outside the vector RNA also had no significant effect on the vector titer. The titer of the vector generated with this two plasmid system is comparable to that obtained using the helper construct pVirPac1.1 containing RRE1, which showed about 5-fold lower enveloping efficiency. Thus, incorporation of RRE-2 into a helper construct not only increased its safety by reducing the possibility of homologous recombination, but also unexpectedly increased the efficiency of the coating.

Example 5

This example tests whether MGMT gene expression from HIV-LTR spliced mRNA is sufficient to efficiently select MGMT-transduced cells by BG and BCNU. Figure 10A shows the vectors used. Figure 10B shows effective cell survival and spread in the presence of cytocidal BG / BCNU concentrations. The designations are as follows: M, MGMT; I, IRES; G, GFP; W, Woodchuck's post-transcriptional element, and C, CMV promoter. All of the above elements were cloned in the pN1 vector, downstream of the RRE element, so that any gene 3 'to the RR element would be expressed from the spliced mRNA, as the splice acceptor site was located adjacent the insertion site of that gene. The figure demonstrates that while control cells not transduced with the lentiviral vector ("CGFP") neither expand nor survive, all vectors expressing MGMT survive and spread.

Cells transduced with pNICMIG were dead after 14 weeks, while cells transduced with pNIMCG were dead after 21 weeks. It is important that the cells transduced with <RTIgt; 0 </RTI> 0 * 0.000 with pNIMIG and pNIMIG-W remained viable for 29 days. weeks, indicating long-term survival of cells transduced with the vector without the inserted internal promoter.

Surprisingly, vectors expressing MGMT from RNA spliced to HIV-LTR were very efficiently selected. Expression of MGMT does not limit the extent of gene burden to either MGMT or selection genes. These results indicate that any gene can be expressed by the HIV-LTR promoter in T cells. Similar data was obtained for non-T cells, demonstrating that expression of mRNA genes spliced to HIV-LTR can be used to express genes of many cell types.

In addition, the two-cistron nature of the "MIG" constructs demonstrates that multiple genes can be expressed when bound by IRES. Examples in addition to the above include MGMT or other selectable genes with a chemokine, interferon, or other genetic antiviral agent.

Example 6

Human CD4 + T cells were tested for their sensitivity to BG and BCNU. CD14 + cells depleted of CD14 were purified from peripheral blood with or without cytokine mobilization. These cells were cultured in 3 µg / ml PHA and 5 ng / ml IL-2 for 5-7 days prior to BG and BCNU treatment. Under appropriate conditions in the absence of BFG and BCNU, CD4 + cells can be expanded up to 100-fold in 10 days in culture. The doses tested were 0; 0.2; 0.5; 2; 5; 10; 20; 30 and 40 μΜ BG and 0,2, 5,10,20, 30 and 40 μΜ BCNU. A series of three experiments was performed.

Figure 11 shows representative results of the above tests. Propagation was reduced to 20 fold by 10 μΜ BG and μΜ BCNU and to about 10 fold or less at 20 μΜ or more BCNU. Treatment of CD4 + BCNU cells did not result in growth delay. Thus, unlike the SupT1 cells used in the above example, CD4 + cells are not susceptible to low dose BCNU alone. This suggests greater MGMT expression of primary CD4 + cells, although variability in MGMT expression based on variability among T cell donors is expected.

The presence of both BG and BCNU, both 10 μΜ or greater, was best for CD4 + cell sensitization, while the same cell sensitization was achieved at 0.5 μΜ BG and 15 μΜ or more BCNU. Increasing the dose of BG did not increase the number of cells killed, while increasing the dose of BCNU increased the toxicity to CD4 + cells.

As shown in Figure 11, the level of selection generally increased 5-fold from a baseline of 20% to 100%. In one experiment, the selection increased from 3% to more than 90%. Po φ «·· • * ·· φ · φφ ΦΦΦΦ

Φ ΦΦΦ φ

φ ► ♦ φ φ selection and 10 to 100 fold multiplication was observed one week; multiplication 20 to

400-fold was observed at 2.5-fold selection, with an average of 80-fold multiplication.

Example 7

Previous work suggests a correlation between cleansing bone marrow autoantrants with improved disease-free survival after autologous transplantation. Removal of normal T cells from the allograft was also used to reduce the risk of immune responses between the host and the transplant. Figure 13A demonstrates the efficiency of transducing tumor cells in a single transduction cycle using pN1 (cPT) ASenvGFP. Figure 13B confirms that tumor cells can be selectively and efficiently killed as opposed to CD4 + stem cells since the latter do not show significant transduction after one cycle of transduction.

The application of the above to the removal of tumor cells from the autologous bone marrow graft begins with the cultivation of bone marrow or the collection of peripheral blood stem cells. CD34 + cells are purified on a CD34 antibody column and then transduced with a conditionally replicating vector at an MOI of 2 to 400 for 2 to 16 hours. The cells are optionally incubated in the presence of one or more activating factors, such as cytokines, for either assisted transduction or maintenance of these cells. Thereafter, the cells are transferred to the subject to be treated.

The ability to similarly interfere with T cells in a foreign graft uses conditionally replicating vectors in combination with a highly efficient, stable transduction protocol, similar to US Patent Application Number (to be assigned) filed on August 31, 2000, below. No. 272 000 400, for selective T cell transduction.

Example 8

Figure 14, panel B, shows the effective pseudotyping of the HIV-1 vectors of the invention with various envelope proteins.

The same original titers in a lentiviral vector, pseudotyped with the VSV-G envelope protein, can be effectively increased by either high-speed centrifugation or diafiltration. The vector pN1 (cPT2) ASenvGFP was coated with pVirPac1.2Rz2. The following table shows (a) the total protein in the sample, (b) the titer of the vector in the sample, and (c) the degree of purification of the vector from foreign material in the sample.

»· ♦♦ 999 · · ♦♦ 999

9 9

9 9

9 9 9

* 9

99

Process step Total protein, mg Vector titer, TU / ml Degree of cleaning (multiple) Start after clearing 3207 5.9 x 10 ° ON. Concentration 722 8.2 x 10 ' 61.7 Diafiltration 224 6.8 x 10 ⁸ 1650

As can be seen from the table, the total protein content of the sample supernatant of the vector after clarification from cell debris is 3027 mg, while the vector titer at this stage is 5.9 x 10 ⁶ . The sample is then concentrated by ultrafiltration, the total protein content of the sample being 722 mg and the vector titer increased to 8.2 x 10 ⁷ , at a 61.7-fold purification degree. After concentration by ultrafiltration, the sample is diafiltered, further reducing the total protein content of the sample to 224 mg, increasing the vector titer to 6.8 x 10 ⁸ per ml and 1650 times the degree of purification. This demonstrates that non-vector foreign proteins can be removed and the lentiviral vector that is pseudotyped with VSV-G can be purified as described above.

The above facts can be utilized in conjunction with vectors produced in Hela-tat cell lines giving 10 ¹⁰ titers as shown in Figure 16.

Example 9

Alternative helper constructs for pseudotyping conditionally replicating vectors were developed by placing VSV-G expression under Rev-dependent control. Since RRE-containing mRNAs are not expressed in the absence of Rev, they have been postulated to be defective due to the presence of cis repressive sequences (CRS) located in the gag / pol, pol, and env genes. CRS has been reported to capture mRNA in the nucleus, destabilize mRNA, or prevent mRNA from associating with ribosomes. Thus, the presence of CRS and Rev, located in trans, combined with mRNA splicing can be used to regulate gene expression.

The Rev / RRE / CRS system was used to control VSV-G expression in a Rev-dependent manner. Helper constructs for this approach include a 5 'splice donor site; HIV-2 RRE and 3 'splice acceptor site (preferably for tat / rev); A CRS fragment from the HIV-2 pol region to reduce the likelihood of recombination with HIV-1-based vectors, and optionally a non-inducible promoter, such as the CNV immediate early (IE) promoter, instead of any inducible promoters. The RNA encoding VSV-G, although produced without induction, is still regulated by the presence of Rev, since only the uncut RNA can be exported and expressed in the cytoplasm without Rev. VSV-G expression is still mediated by Rev because Rev counteracts the retention of transcripts in the nucleus, controlled by mRNA splicing and CRS.

<Toto toto toto toto 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 toto 9 to to 9 9 *

Preferably, the donor site is not a synthetic 5 'β-globin splice donor site, but a splice donor site of native HIV or an HIV analog. The HIV-2 RRE is a 280 bp fragment containing both the HIV-2 RRE and the tat / Rev splice acceptor site and the CRS is a 550 bp internal fragment of the HIV-2 sequence of the CRS and RRE elements from HIV-2 can be prepared using PCR. Non-limiting examples of such helper constructs include pCGCSRRE, pCGCRSRzRRE and others shown in Figures 15A to 15E. The final plasmid constructs were confirmed by multiple digestion with restriction enzymes. While pEH-GP and pCMV-GP are constructs that constitutively express a gag-pol polyprotein, other promoters, including a lentiviral or HIV promoter, may be used if desired.

Figure 17 shows the results of the use of many different combinations of splice donors for VSV-G expression dependent on Rev. As shown, the removal of tetracycline results in Rev-dependent expression.

Further examples of combinations of splice donor sites, as well as recognition sequences, are provided below. While all may be used, HIV major, HIV-1 env, HIV-2 major and analogous combinations of splice donor sites are preferred.

STANDARD EDITING DONOR: BETA-GLOBIN EDITING DONOR: HIV MAJOR EDITING DONOR:

HIV-1 ENV EDITOR DONOR:

HIV-2 ENV EDITOR DONOR:

HIV-2 MAJOR EDITOR DONOR: ANALOGUE EDITOR DONOR:

NNNNAGGTAAGTNNN (SEQ ID NO: 7) NGGGCAGGTAAGTAT (SEQ ID NO: 8) NNGACTGGTGAGTAN (SEQ ID NO: 9) AAAGCAGTAAGTAGT (SEQ ID NO: 10) AGACAAGTGAGTAAG (SEQ ID NO: 11) NNGAAGGTAAGTGC NO: 13)

Example 10

Various storage buffers and conditions were tested to determine suitable storage conditions for the vectors before use. The results are shown in Figure 18. The ability to store vectors at -20 ° C and in pharmaceutically acceptable buffers without causing significant losses in vector recovery has a tremendous advantage in maintaining the vector before use. Of course, storage can also be readily performed at about -80 ° C, about -20 ° C, and about 4 ° C in other pharmaceutically acceptable compositions.

• 94 · 94 · 94 * 94 94 * 4 · 4 4 ♦ 94 4 «4 4 44 44

4 ·

4 «« 3 4

4 49 49 • 4 ««

They did

This example describes the amino acid sequence of a chimeric HIV CTL epitope for use in practice. This sequence (SEQ ID NO: 18) contains the first translation initiation methionine (M), followed by various contiguous sub-sequences corresponding to p17, p24, p15, Pol, Rev, gp120env, gp41env, respectively. nef.

MKIRLRPGGNKKYKLKHIVWASRELERFGSEELRSLYNTVAVLYCVHQ

KIEVKDTKEALDTENRNQESQNYPIVQNLGQMVHQALSPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATP

QDLNTMLNTVGGHQAAMQMLKATINEEAAEWDRLHPVHAGPIAPGQ

MREPRGTSTLQEQIAWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSI

FRDYVDRFYKTLRAEQATQEVKNWMTETLLVQNANPDCKTILKALL

EDMMTACQGVGGPGHKARLVQEGHQMTCDCTERQANFGNFPQSRLEPTAPPEITLWQRPLVDTVLEDMNLVLVGPTPVNISPIETVPVKLGPKVKQWPLA

LVEICTEMEKEGKISKIGPTVLDVGDAYFSVPLDEDFRKYTAFTIPSIW

KGSPAIFQSSMTINPDIVIYQYMDDLYVDLEEGQHRTKIEELRQHLLR

WGFTTPDKKPIKLPEKESWLVGKLNWASQIYAGIKVKQLIITEEAELE

ILKEPVHGVYQIYQEPFKNLKTGDVKQLTEAVKJTTESIVIWPIQKETW

ETWWTEYWPLVKLWYQLEPIVGAETFYVDGAANKALQDSGLEVNIV

TDSQYALGIESELVSQUEQLLAWVPAHKGYEEAjEVIETAYFILKLLLW

KGEGAVISGWILNTYRVKGIRKNYAENLWVTVYYGVPVWKEATTTLFCASDAKAYDPNPQE

WLHEDIISLWDQSLKKLTPLCVTLNCSFNVTTLINTSYTLINCKSSTIT

QACPKCKNVSTVQCRPWSTQLLLNGSLAEEDIVSIEINCTRPNNNTRK

KITLGPGRVLYTTGENNTLKQIVEKLREIKQFKPEIVMHSFNCGGEFFY «<· ·«

CNSTQLFLPCRIKQIINRWQEVGKAMYAPPIEGQIRCLSNITGVKIEPLG

VAPTKAKRRWQRRAIEAQQHLGIKQLQARVLAVERYLKDQQLLGITTVPWNASWWYIKI

FIMIVGVLSIVNRVRQGYSPLSFQTHRLVDGFLTLLYHRLIDLLLIAKR

GRRGWAALKYSLLNATAIAVDRVIEIVQRTCRAILHIPRRIRQGLERAL

LWPAIRERMVGFPVRPQVPLRPMTYKAAHDLSHFLKEKGGLEGLIYSQ

KRQDILDLWVYHTQGFFPDWQNYTPGPGTRYPLCFGWCFKLVPVVL

MWKFDSKLAFHHVARELHPEYFKDCPexample 12

This example provides a summary of embodiments of the invention as described above. The invention encompasses a conditionally replicating viral vector comprising a gene (or other nucleic acid) to be expressed and one or more first nucleotide sequences, wherein the vector (a) replicates in the host cell only when complementing with a wild-type virus strain a helper virus or helper vector, each of which is susceptible to the presence of said one or more first nucleotide sequences; (b) is resistant to the presence of said one or more first nucleotide sequences; and (c) comprises one or more substitutions, insertions, or deletions that reduce the likelihood of a replication competent vector being formed.

To reduce the likelihood of a replication-competent vector, the invention provides vectors that contain sequences degenerate with respect to one or more helper construct sequences, host cell nucleic acid content, or other nucleic acids. In addition, helper constructs for use in conjunction with these vectors for packaging. These helper constructs may also contain sequences degenerate with respect to one or more vector sequences, host cell nucleic acid content, or other nucleic acids.

The vectors of the invention may also contain one or more sequences that target a helper construct. These sequences comprise genetic antiviral agents and are preferably (or encode) ribozymes or antisense nucleic acids. Preferred ribozymes and antisense sequences are capable of pairing based on base complementarity with the respective targets by

«· Φ φ φ φ φ φ · · · · · · · · · · · · φ φ φ φ φ φ φ

G-U base pairing, except for standard base pairing A-T, A-U, and G-C. The helper constructs of the invention may likewise also contain sequences of the same type for targeting the vector.

The helper vectors of the invention may also contain one or more sequences that affect the location or cellular transport of the RNA expressed by the helper as compared to the vector. For example, the helper vectors may comprise an intron, a heterologous RRE relative to the vector, or a heterologous gag sequence relative to the vector. Helper vectors may also contain degenerate gag and / or pol sequence (s) as compared to the vector, such that co-localization of these helper and vector RNAs is less likely. Helper vectors may also contain splice donor sites so that the packaging (and RRE) signals (sequences) are removed from the RNA to prevent their co-localization with the vector RNAs. These splice donor sites may also be present in helper vectors to effect different vector transport compared to the helper vector or wild-type viruses.

Helper vectors of the invention may also be capable of pseudotyping the vectors of the invention. Exemplary examples include VSV-G, RDI 14, rabies virus, GALV, and chimeric envelope proteins. The helper vectors of the invention are also preferably derived from a virus heterologous to the vector. An example is helper vectors derived from HIV-2. The methods of the invention include the use of these helper vectors for the replication of the vectors of the invention. Exemplary vectors of the invention include the pN1 and pN2 series shown in Figures 1A through 1K, particularly with removal or substitution of GFP sequences. Preferably, these vectors retain at least an antisense or cPT-containing sequence. Exemplary helper constructs of the invention include the pVirPac series as shown in Figure 6A to 6G, with or without ribozymes.

All vectors and helpers of the invention may be degenerated as described above or in other parts of the constructs, if necessary. Preferred degeneration is relative to the codons, preferably used in human cells. These vectors and helpers may also be chimeric in nature, so that they are derived from a variety of naturally occurring as well as synthetic nucleic acids. Preferred chimeric constructs contain both HIV-1 and HIV-2 sequences. However, particularly preferred vectors are derived from HIV-1 or encode an HIV-1 Tat protein. Alternatively, these vectors do not encode a Tat protein, but this is delivered by a helper vector, a wild-type virus, or a host cell.

Also, the vectors of the invention preferably do not encode or express viral accessory proteins. These vectors may also contain a nucleic acid that reduces, minimizes, or prevents viral infection when present in, or delivers to, the host cell.

9999

9 9 • · 999 ·· ·· Resistance to toxic agents. Examples of such nucleic acids include cell surface protein or incomplete CCR5 protein, preferably CCR5A32T protein. Other examples include nucleic acids encoding a drug or DNA damaging agent, preferably a multispecific drug resistance, or a MGMT variant, particularly a G156A variant. Other examples include nucleic acids encoding a transdominant phenotype. An exemplary example is the transdominant HIV-1 gag sequence.

The vectors of the invention may also contain sequences to aid expression of the heterologous sequence. An exemplary example is the isolation sequence. The vectors may also contain a sequence that enhances transduction of the vector into cells. An exemplary example is the 545 base pair sequence containing cPT described herein, although smaller or larger fragments containing cPT may also be used. The vectors may also contain LTRs for the expression of genes, as well as nucleic acids, including LTRs, modified at the transcription binding sites described herein. These modified LTRs are preferably contained in lentiviral vectors. The methods of the invention include the use of these vectors.

The vectors of the invention may also express at least two genes using suitably positioned splice sites or IRES elements. The vectors may also be packaged into viral particles with one or more chimeric envelope proteins that stimulate target or host cells for transduction. In addition, the vectors may be packaged in cells where the helper is a protein or the helper comprises a Rev / RRE / CRS system for regulating gene expression. The methods of the invention include the use of such vectors.

The vectors of the invention may also be used to selectively kill transduced cells in conjunction with an administered agent that is not normally toxic to non-transduced cells. It is preferred that these vectors act through the production of a nucleic acid molecule rather than a protein. An exemplary example is the production of an anti-MGMT antisense or ribozyme nucleic acid molecule. The methods of the invention include the use of such vectors.

The invention also provides methods for storing vectors of the invention.

All references cited herein, including patents, patent applications, and publications, are incorporated herein by reference in their entirety. As used herein, all technical terms presented herein in the singular are intended to include both the singular and the plural.

While the present invention has been described herein with reference to preferred embodiments thereof, it will be apparent to those skilled in the art that in these preferred embodiments, it is possible to:

The invention may be practiced otherwise than as specifically described herein. The present invention is intended to include these changes and other embodiments. Accordingly, the invention includes all modifications included within the spirit and scope of the invention as defined in the appended claims.

Claims (22)

PATENT CLAIMS A conditionally replicating viral vector, comprising the gene to be expressed and one or more first nucleotide sequences, wherein the vector: (a) replicate in the host cell only when complemented by a wild-type virus strain, helper virus or helper vector, each of which is susceptible to the presence of said one or more first nucleotide sequences; (b) is resistant to the presence of said one or more first nucleotide sequences; and (c) comprises one or more substitutions, insertions, or deletions that reduce the likelihood of a replication competent vector being formed. The vector of claim 1, wherein said one or more substitutions is a codon degeneration, preferably used in human cells. The vector of claim 1, wherein the vector is a chimeric vector comprising the sequences of HIV-1 and HIV-2. The vector of claim 1, wherein the vector does not encode or express viral accessory proteins. The vector of claim 1, wherein the vector is derived from HIV-1 or encodes an HIV-1 Tat protein. The vector of claim 1, wherein the one or more first nucleotide sequences is one or more ribozyme or antisense sequences. The vector of claim 1, wherein the vector does not encode or express the Tat protein. The vector of claim 1, further comprising a nucleic acid sequence that diminishes, minimizes, or prevents viral infection when present in the host cell or renders the cell resistant to toxic agents. ···· ··· 9 999 994 994 994 994 994 994 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 The vector of claim 1, wherein said first nucleotide sequence encodes a transdominant phenotype. The vector of claim 1, wherein the vector further comprises an insulator sequence. The vector of claim 1, further comprising an LTR or a modified LTR for regulating the expression of the nucleic acid sequence present in said vector. The vector of claim 1, further comprising HIV splice sites or internal IRES. A helper vector that is capable of complementing a vector according to any one of the preceding claims. The helper vector of claim 11, wherein the helper vector is further capable of pseudotyping said conditionally replicating vector. The helper vector of claim 11, further comprising one or more ribozymes or antisense sequences targeted to said conditionally replicating vector. The helper vector of claim 11, wherein the vector is derived from HIV-2. A vector according to any one of claims 1 to 12, packaged in a viral particle comprising one or more chimeric envelope proteins. A helper capable of complementing the vector of any one of claims 1 to 12, wherein the helper is a protein. 19. A method of expressing one or more nucleic acid of interest, characterized in that a vector according to any one of claims 1 to 12 or 17 is used. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · A vector production method according to any one of claims 1 to 12 or 17, characterized in that high-speed centrifugation and no ultracentrifogation are used. A method of killing cells, wherein the cells are transduced with a vector according to any one of claims 1 to 12 or 17. A method for selecting hematopoietic stem cells, comprising contacting a population of cells comprising hematopoietic stem cells with a cytotoxic agent after contacting the vector with a vector according to any one of claims 1 to 12 or 17.