Spleen necrosis virus-based vector delivery of anti-HIV-1 genes potently protects human hematopoietic cells from HIV-1 infection.

In this study, we report on the efficacy of using a spleen necrosis virus (SNV)-based vector delivery system to block human immunodeficiency virus type I (HIV-1) replication in human hematopoietic cells. These efforts were directed towards the development of human immune system cell resistance to HIV-1 infection, based on the strategy of "intracellular immunization" via generation of a series of anti-HIV-1 therapeutic constructs carrying scFvs, single-chain variable fragments, against HIV-1 integrase and reverse transcriptase in combination with the trans-dominant mutant of HIV-1 Rev, RevM10. The efficiency of the anti-HIV-1 constructs were tested in viral challenge assays with different doses of HIV-1 NL4-3, Bal, 89.6 and R7-GFP strains. These experiments demonstrated the reduction of HIV-1 replication by these retroviral vector constructs in a range of 4- to 10-fold in CD4+ T-lymphocytes, human peripheral blood mononuclear cells (PBMCs), and primary human macrophages. We observed selective efficiency of SNV-based therapeutics in H9, C8166 and Jurkat T-lymphocytic cell lines, demonstrating the most efficient inhibition of HIV-1 replication in Jurkat T-cells. Thus, these data are the first demonstration of the ability of SNV-based retroviral vectors with select transgenes, which may have certain molecular advantages over other retroviral vector systems, to combat HIV-1 replication in human hematopoietic cells and support the potential for using SNV-expressed constructs in anti-HIV-1 molecular therapeutics.

Cross-packaging of human immunodeficiency virus type 1 vector RNA by spleen necrosis virus proteins: construction of a new generation of spleen necrosis virus-derived retroviral vectors.

The ability of the nonlentiviral retrovirus spleen necrosis virus (SNV) to cross-package the genomic RNA of the distantly related human immunodeficiency virus type 1 (HIV-1) and vice versa was analyzed. Such a model may allow us to further study HIV-1 replication and pathogenesis, as well as to develop safe gene therapy vectors. Our results suggest that SNV can cross-package HIV-1 genomic RNA but with lower efficiency than HIV-1 proteins. However, HIV-1-specific proteins were unable to cross-package SNV RNA. We also constructed SNV-based gag-pol chimeric variants by replacing the SNV integrase with the HIV-1 integrase, based on multiple sequence alignments and domain analyses. These analyses revealed that there are conserved domains in all retroviral integrase open reading frames (orf), despite the divergence in the primary sequences. The transcomplementation assays suggested that SNV proteins recognized one of the chimeric variants. This demonstrated that HIV-1 integrase is functional in the SNV gag-pol orf with a lower transduction efficiency, utilizing homologous (SNV) RNA, as well as the heterologous vector RNA of HIV-1. These findings suggest that homology in the conserved sequences of the integrase protein may not be fully competent in the replacement of protein(s) from one retrovirus to another, and there are likely several other factors involved in each of the steps related to replication, integration, and infection. However, further studies to dissect the gag-pol region will be critical for understanding the mechanisms involved in the cleavage of reverse transcriptase, RNase H, and integrase. These studies should provide further insight into the design and development of novel molecular approaches to block HIV-1 replication and to construct a new generation of SNV-based vectors.

Spliced spleen necrosis virus vector RNA is not encapsidated: implications for retroviral replication and vector design.

RNA splicing is a complex event in the retroviral life cycle and can involve multiple steps, as well as cis-acting sequences, to maintain a proper balance of spliced and unspliced viral RNA for translation and encapsidation. The retroviral RNA can be processed by cellular machinery and enables the removal of intronic sequences. We aimed to utilize the removal of a synthetic intron for targeted gene expression. To analyze intron removal and gene expression, we have constructed a novel self-inactivating gene-activating (SIGA) vector for potential universal gene therapy. New vectors for gene therapy are necessary for safe and effective gene delivery in humans. The SIGA vector is derived from spleen necrosis virus (SNV), which is an avian reticuloendotheliosis virus. The vector was designed so that expression of a therapeutic gene is blocked in helper cell lines due to an intervening sequence containing various blocks in transcription and translation. However, after one round of retroviral replication, the intervening sequence should be removed by the cellular machinery and the therapeutic gene will be selectively expressed in target cells. Our studies show that the intervening sequence in SIGA vector RNA is partially spliced. However, spliced vector RNA was not transduced to target cells. Previous studies showed that an infectious SNV vector enabled transduction of spliced RNA. However, yet-undefined differences in infectious and replication-deficient retroviral replication may have an effect on the transduction of spliced RNA. The results of this study present key information on spliced RNA and its encapsidation, as well as data for the construction of a new generation of SNV-derived retroviral vectors.

Cell-type-specific gene delivery into neuronal cells in vitro and in vivo.

The avian retroviruses reticuloendotheliosis virus strain A (REV-A) and spleen necrosis virus (SNV) are not naturally infectious in human cells. However, REV-A-derived viral vectors efficiently infect human cells when they are pseudotyped with envelope proteins displaying targeting ligands specific for human cell-surface receptors. Here we report that vectors containing the gag region of REV-A and pol of SNV can be pseudotyped with the envelope protein of vesicular stomatitis virus (VSV) and the glycoproteins of different rabies virus (RV) strains. Vectors pseudotyped with the envelope protein of the highly neurotropic RV strain CVS-N2c facilitated cell type-specific gene delivery into mouse and human neurons, but did not infect other human cell types. Moreover, when such vector particles were injected into the brain of newborn mice, only neuronal cells were infected in vivo. Cell-type-specific gene delivery into neurons may present quite specific gene therapy approaches for many degenerative diseases of the brain.

The history and principles of retroviral vectors.

Retrovirus-derived gene transfer systems (retroviral vectors) are the most commonly used gene transfer tools in modern biology. They have been used to study various aspects of retroviral replication, the organization and function of oncogenes and other eucaryotic genes, and, recently, to transduce therapeutic genes to cure inborn errors of metabolism, cancer, AIDS, and many other diseases in man. Highly oncogenic retroviruses served as a model for the construction of artificial retroviral gene transfer systems. These viruses carry a non-viral gene in their genome in addition or substituting for viral protein coding sequences. The replication of such defective retroviruses depends on the presence of a wild-type-virus, which supplies all proteins in trans for particle assembly and infection of a new target cell. Thus, highly oncogenic retroviruses can be considered as naturally occurring gene transfer vectors. Following this principle, cell lines have been constructed which express retroviral protein coding sequences from plasmid DNAs and which contain a viral genome in which the protein coding sequences have been replaced with a gene of interest. This article describes the history, principles, and basic building blocks of first and modern retrovirus-derived gene transfer systems.

Reticuloendotheliosis viruses and derived vectors for human gene therapy.

The reticuloendotheliosis viruses (REV) spleen necrosis virus (SNV) and reticuloendotheliosis virus strain-A (REV-A) are amphotropic retroviruses which infect a large variety of cells of avian and some mammalian species. They normally do not infect primate or rodent cells. However, they efficiently infect and integrate their genome into that of human cells when they are pseudotyped with the envelope protein of other mammalian retroviruses or the G protein of vesicular stomatitis virus (VSV) or rabies viruses (RV). Moreover, SNV-derived retroviral vectors, which display single chain antibodies or other targeting ligands on the viral surface enable cell-type-specific gene delivery into various human cells. My laboratory has developed genetically engineered REV vectors, which are capable of infecting non-dividing cells such as quiescent human T-cells, primary monocyte-derived macrophages, and mature neurons. Thus, REV-derived vectors appear to be very interesting candidates for the further development of vectors for human gene therapy. This article reviews the replication of REVs and vectors derived from REV-A and SNV for gene transfer into human cells.

Retroviral packaging cells encapsulated in TheraCyte immunoisolation devices enable long-term in vivo gene delivery.

The method of delivering a therapeutic gene into a patient is still one of the major obstacles towards successful human gene therapy. Here we describe a novel gene delivery approach using TheraCyte immunoisolation devices. Retroviral vector producing cells, derived from the avian retrovirus spleen necrosis virus, SNV, were encapsulated in TheraCyte devices and tested for the release of retroviral vectors. In vitro experiments show that such devices release infectious retroviral vectors into the tissue culture medium for up to 4 months. When such devices were implanted subcutaneously in SCID mice, infectious virus was released into the blood stream. There, the vectors were transported to and infected tumors, which had been induced by subcutaneous injection of tissue culture cells. Thus, this novel concept of a continuous, long-term gene delivery may constitute an attractive approach for future in vivo human gene therapy.

Replication of lentiviruses.

Lentiviruses belong to a subfamily of the retroviruses usually associated with persistent infections in animals and humans. They have complex replication cycles involving numerous regulatory and accessory proteins, which sets them apart from the oncoretroviruses and spumaviruses, the two other main subfamilies of the retroviruses. Studies over the years have elucidated the various molecular mechanisms involved in the replication of lentiviruses. The first step involves the fusion of the envelope glycoprotein (gp120) to the host cell membrane followed by entry of the virus into the host cell. Immediately following viral entry is reverse transcription, integration, gene expression, encapsidation, budding and lastly virus maturation. This review focuses on the molecular mechanisms involved in the lentiviral replication, using human immunodeficiency virus type I (HIV-1) as an example.