GB2543730A - Use of replication competent vector to eradicate viral latency presented on human immunodeficiency virus - Google Patents

Abstract

A modified human immunodeficiency virus genome vector is disclosed characterised in that the vector is replication-incompetent in a living cell which is free from the wt-HIV virus, but replication-competent in a cell infected with the wild-type HIV virus, due to the wild-type virus providing the mutant virus with viral components essential for viral replication, which are mutated in the modified virus. Nucleic acids in the modified HIV vector may encode mutated viral components such as reverse transcriptase, integrase, proteases, matrix and capsid proteins, infectivity and regulatory factors or viral nucleic acids. Preferably, the modified HIV vector can be used to treat or prevent an infection caused by any genetic variant of HIV. Preferably, the conditionally replicative HIV vector eradicates wild-type HIV-positive (HIV+) cells by promoting host cell apoptosis and suppressing the assembly and/or maturation of the wild-type virus through competition for the aforementioned viral components required for replication. The modified HIV vector is a defective-interfering virus (also called defective-interfering particle, DIP, therapeutic-interfering particle, TIP), may be used to eradicate latent HIV infection and may encode antisense RNAs against viral nucleic acids and/or proteins. Methods of the invention may also be used to treat other viruses.

Description

DESCRIPTION Field of the Invention

This innovation is generally in the area of the treatment of virus infections, more particularly relates to the treatment of acute and latent HIV infections. The method involves administration into living organism viral vector, genetically altered form of HIV virus. In order to work, in the same treated organism, the HIV vector and the HIV should be genetically similar and be able to share the same protein components, building blocks necessary for viral assembly. Based on this example, eradication of HIV infection rely on deleting all HIV infected cells, using viral vector which in HIV positive cells and as a result of the presence of wild-type HIV proteins allows such vector to maturate and become a replication-competent. Consequently, released assembled HIV particles containing the vector's RNA will further spread on other cells, continuously replicate releasing more vector infectious particles and decreases the wild-type HIV level. The process by which the vector will decrease the wild-type HIV level are combination of following events beginning from apoptosis of all infected cells having the vector insert and at the same time stopping of formation of new latently infected cells as a result of vector modification within the LTR5' promoter. Additionally, increased vector replication rate compared with wild-type HIV will results in hijacking viral protein components produced only by the wild-type HIV and attaching them during packaging process to the vector's unspliced RNA. The wild-type HIV maturation would be suppressed by the abundant vector replication and the wild-type HIV assembly would be suppressed due to lack of access of the protein produced by wild-type HIV and not presented in vector RNA sequence but utilized mainly by the vector during assembly process within the cell cytoplasm.

Background of the Invention

Current medicine is capable to control virus infection relatively good. Combination antiretroviral therapy suppresses but does not eradicate any subtype of human immunodeficiency virus in infected persons. Low-level viremia can be detected despite years of suppressive antiretroviral therapy which only delays progression but can't eliminate the source of disease. An antiretroviral agent inhibit and interrupt different parts of the viral life cycle but are unable to get rid of the latent form of the virus itself. The existence of a latent viral DNA sequences integrated into cell genomic material is a current main problem which causes that virus re-emerges rapidly after antiviral treatment is stopped. It has been known for long that reverse transcriptase is necessary in HIV life cycle. This enzyme is required to generate complementary DNA strand from an RNA template, a process termed reverse transcription. HIV assembly pathways are a complex and dynamic process, involving many steps. All viral components needs to be traffic from their point of synthesis to site of assembly on the plasma membrane. All viral gene products are encoded on the genomic RNA, which also serve as mRNA for Gag and Gag-Pro-Pol, whereas singly or multiply spliced RNAs are translated to produce Env and accessory proteins. HIV virus depends on both cellular and viral factors for efficient transcription of its genome. The proteins that make up the retroviral core particle are translated as part of polyprotein precursors. Processing of these precursors is accomplished by a viral protease (PR) that is contained within one of the precursors. During virus assembly, the processing sites within the retroviral precursor proteins are cleaved by the viral PR. Accurate and precise PR-mediated precursor processing is an absolute requirement for the production of fully infectious viral particles; mutations that produce imprecise cleavage at individual sites or that alter the order in which sites are cleaved result in the elaboration of aberrantly assembled virions that are markedly less infectious. Furthermore, reductions in PR activity can have pleiotropic effects because the PR is responsible for cleaving the Gag-Pro-Pol precursor to generate active reverse transcriptase (RT) and integrase (IN). Maturation of virus progeny occurs by budding and envelopment of the filamentous helical nucleocapsids at the cell surface. The process of RT cleavage event during HIV assembly doesn't yet fully understand that's why potentially upholds the right of this innovation to use process of viral competing for eradication of viral infection based on RT viral enzyme. Assembly, release and maturation of HIV particles comprise a highly dynamic sequence of events, characterized by a series of dynamic rearrangements of the viral structural proteins and overall virion architecture. HIV morphogenesis is a relatively rapid and asynchronous process, showing high variability between cells and individual virons. It is known that Gag polyprotein traffics to the plasma membrane of the host cell and also recruits other viron components for example host cell ESCORT machinery, needed for virus abscission. However, the release of virons by budding, from the cell membrane are non-infectious particles. They needs to go through radical morphological rearmament leading to formation of mature capsid formation.

Summary of Innovation.

The method described herein uses genetically modified HIV virus to eradicate HIV latency. Prior the vector administration, antiretrotheraphy should be stopped, allowing introduced viral vector to replicates and infect other cells. In order to deliver the vector genome into the cell, such vector have to be packaged together with all HIV proteins necessary to insert the vector RNA into the cell genome. As opposite, if the vector genome would get inserted into HIV-free cell, it life cycle would ends as having missing or altered at least one building block within it genome, would prevent the vector from assembly into infectious particle. However, if the vector would be introduced into HIVpositive cell, the vector would be able to maturate as the missing protein necessary to create either the HIV core, envelope or proteins binding the unspliced viral RNA, could be obtained from wild-type HIV. The modification to design a replication-incompetent in HIV negative cells and replication-competent in HIV positive cells may apply to any HIV viral protein presented within HIV. Such missing protein within the vector would not allow to maturate the vector particle into replication-competent virus without the present of wild-type HIV.

For instance, modified HIV vector, having altered or lacking in this example reverse transcriptase (RT) codding sequence causes that modified virus vector is disable to transcribe it viral RNA into DNA. Therefore for the vector to obtain necessary protein (in this example RT) cell must also express missing protein. The process in which gain of the RT would be only possible, if infected with vector cell also express and translate the wild-type HIV viral genome, together with RT protein. It is still unknown exact process of HIV maturation. It is believed that production of unspliced RNA and packaging it together with viral enzymes doesn't necessary have to have the same origin and originates from the same DNA viral genetic sequence. That's why it is possible if two similar strands of HIV viruses are replicates at the same time, competition for same protein such as the RT protein inside same cell can be introduced between vector and HIV.

Increased vector replication which could be achieved by removing for instance the API sequence within the LTR5' promoter, could generate imbalance during transcription between the wild-type HIV and the vector which would result in attachment of majority of the RT protein to mRNA originated from the vector. As a result the wild-type HIV would be left without it and unable to maturate. That would decline extracellular number of wild-type viruses capable to infect other cells. At the same time the number of mutant HIV viruses capable to successfully infect other cell would increase, spread and infect any cells as long as any RT from wild-type virus could be still expressed and possible to uptake. An increased viral replication caused by the vector would lead to apoptosis which will terminate the wild-type HIV and mutant replication in that cell simultaneously. Process will follow until the vector can obtain RT from any remaining wild-type viruses during the assembly process. Once, all wild-type virus production would stop due to host cell apoptosis, the vector replication would also stop and process of elimination of wild-type virus successfully would end up. The body would be free from wild-type of virus. Finally, mutant virus alone would be unable to deliver it genomic sequence, infect other cells and the number of it reservoirs would also decline with time.

Vector characteristics when infects HIV-free cell.

In order to prevent forming new latency and increase replication rate, compared to the wild-type HIV the vector needs to have altered the HIV-1 LTR5' promoter and removed AP-1 region (Duverger et al. 2013). The vector lacking the reverse transcriptase (RT) region would not be able to replicate itself inside the infected cells but only if the wild-type HIV-1 will also be present and can provide the RT. This process occurs because the unspliced mRNA and the virus proteins are transported via different routes and as a result a recombination of genetic material between donating mRNA viruses and gaining can take place. The RT protein is necessary to transcribe the viral RNA into double stranded DNA and without it, the virus cannot integrate its genome into a new host cell. However, during vector construction, the RT would have to be packed in vitro with other HIV-1 proteins. Consequently, this construct will be able to insert its genome (lacking the RT genome sequence and with few other modifications which will be explained later) into CD4 lymphocytes and other cells. As a result, during transcription, this vector will be producing all viral components, but not the RT. At this point, even if the vector would assemble into similarly looking the HIV-1 mature virus (which is rather impossible), its life cycle would end as the missing RT prevents its genome to be transcribed into DNA. Thus, above text, describes the basic characteristic of the integration-defective vector, that is unable to integrate in HIV-1 negative cells in its 2nd life cycle (1st life-cycle is when the vector is packaged in vitro with RT and other proteins and can infect the CD4 cell). The vector transcription and the fact that this vector could not go under latent form would lead to cell death (approximately 2 days after infection) and releasing of vector particles which are unable to infect other cells further. This process applies only to the cells which are non-infected with HIV-1 virus during the vector attachment or replication. If the viral vector binds to the cell, which are HIV infected or simultaneously gets infected with the wild-type HIV-1, thus, the process of vector assembly (packaging) will be altered by the presence of the HIV-1 proteins.

Vector characteristics in cell infected with HIV.

Because the HIV-1 wild-type will produce the RT protein during maturation, thus, its protein could be attached to both unspliced-RNAs. If the transcription rate for the unspliced mRNA wild-type HIV-1 and the vector will be equal, than theoretically 50% of the RT should be attached to each of them. Consequently, there should be approximately a 50% of HIV-1 wild-type having its RT and other 50% to the vector. Thus, the life-cycle of the vector would not finish at its 2nd cycle, as having the RT attached to its genome would transform this vector into a virus able to successfully infect another cell. The number of live-cycles for the vector would be strictly dependent to the presence of RT from the wild-type HIV-1 and the HIV-1 itself.

The 50% reduction in number of wild-type HIV-1 having the RT is an estimate and that would not lead to eradicate new infections or to stop the infection. What is necessary is a vector which would have significant higher replication rate compared to HIV-1 in order to decrease the chances of obtaining the RT to the wild-type HIV (on a single cell level) and decrease its spread. At the same time, if an unspliced viral vector predominates that will lead to increase the number of vectors able to infect other cells in plasma and decrease the level of HIV. This process would leads to cells apoptosis that in return will lead to decrease in number of uninfected CD4 cell and the plasma of the HIV-1 (plasma HIV viruses are very short lived). If the replication of the vector reaches the maximum peak, (without causing new latency) that might result in apoptosis of all available CD4 expressing cell. In result the organism would have to be deficient for a short period of time of the CD4 expressing cells. However, any remaining HIV-1 viruses in a plasma, which are short lived (approximately 0.3 days) at the end point could die before they reach any available CD4 cell or additionally can be challenge with ART. However, this process does not apply to latently infected CD4 cells as they do not express CD4 receptor in a similar level to healthy cells, which protects them from additional HIV-1 infection and reactivation of that virus which would result in short death afterwards.

Elimination of latently infected cells.

The main challenge in curing HIV-1, seems to be no longer in the HIV-1 virus, but the resting cells infected with HIV-1 which transcribes its genome sporadically. To resolve this problem, there is a need to identify all infected cell having the HIV-1 genome in it and erase them, simultaneously preventing new infections and new latency. There are few possibilities in which the vector could be useful and help to resolve this issue.

Firstly, to increase vector transcription and to prevent new latency the vector would have to have removed the API region in LTR5' (Duverger et al. 2013). Studies showed that if the region gets removed, it prevents forming new latency almost completely in HIV-l-E subtype (Duverger et al. 2013). Theoretically, that would allow to reduce the CD4 life span to approximately 2.2 days to any CD4 cell infected by this vector (infected or non-infected previously by HIV-1). However, this process could be further improved if our vector would simultaneously express increased level of microRNA such as miR-H3-5P and miR-H3-3P (Zhang et al. 2014). These sequences can be inserted in the removed RT region of the vector and that would increase the viral transcription. Additionally, there are other possibilities and as shown just recently even decreased level of the miR-29a which again could be applied by insertion of complementary RNA in the vector could keep the viral replication active (Patel et al. 2014).

Most importantly as the success of this method relies partly from deprivation of the RT from the wild-type HIV-1 it is very important to keep it replication on a lower level. As the exact number of the mature RT protein which are available to be attached to the HIV could exceed the number of unspliced HIV-1 RNAs (to which the RT is attached during maturation). Thus additional modification in vector could be applied in which it would lower the RT translation and reduce the HIV-1 maturation. This can be obtained by insertion of complementary RNA sequence to the RT-mRNA.

The small interfering RNA sequence could be inserted inside the vector and significantly reduce the RT maturation and accessibility. That in return would reduce the number of HIV-1 competent viruses released during CD4 replication, apoptosis or phagocytosis.

It is important to underline that productively infected an activated CD4 lymphocyte were estimated to have, on average, a life-span of 2.2 days (half-life tl/2 =1.6 days), and plasma virions were estimated to have, on average, a life-span of 0.3 days (tl/2= o.24 days). The minimum duration of the HIV-1 life cycle in vivo is 1.2 days on average, and the average HIV-1 generation time —defined as the time from release of a virion until it infects another cell and causes the release of a new generation of viral particles—is 2.6 days (Perelson et al., 1996). However, the mean half-life of the latent reservoir in patients with virus level undetectable showed to be approximately 43.9 months (Finzi et al. 1999). This process is strictly correlated with the lacking of the expression of CD4 receptor which is dramatically downregulated in latently infected CD4 cells (Kim et al. 2011). That in return disables new HIV attachments to the CD4 and prevents the HIV reactivation, which in normal circumstances would lead the CD4 infected cells to undergo apoptosis. However, to infect these cells, the vector might have find difficulties due to decreased number of CD4 receptors. However this can be overcome using late chemical agents which until now showed to be ineffective such us the IL-7 (Chomont et al. 2009). Studies showed that reactivation of latency is possible in vitro using 20ng/ml of phorbol myristate acetate (PMA)(Kim et al, 2011) or tricostatin A (Suzuki et al., 2013) but there are also many other methods.

Recombination in HIV.

Since two RNA molecules are packaged in each virion, RT may switch from one template to another during reverse transcription. If two RNAs with sequence differences are copackaged in one virion, a mosaic HIV-1 genome containing genetic information from both RNAs could be generated, yielding novel viral genomes (Jetzl et al., 2000). To prevent that, the vector and the virus have to have non-complementary dimer initiation site (DIS). Incompatibility between DIS elements (6-nt sequence) among different strands impairs copackaging of the two genomes (Dirac et al., 2002). The importance of the DIS in recombination is supported by the observation that even within different HIV-1 subtypes, the lack of a homologous DIS presents a strong barrier to recombination, at least in vitro systems (Chin et al., 2007). However, addition of another DIS element inside the HIV-1 (Nikolaitchik et al., 2013) showed in forming a self-dimers with only one packaged copy of RNA. Therefore, HIV-1 can bypass the requirement for packaging two copies of RNA by recognizing a single copy of RNA containing a dimeric structure. HIV-1 regulates RNA packaging by recognizing a dimeric RNA, which leads to the packaging of two copies of the wild-type, full-length viral genome (Nikolaitchik et al., 2013). This could be used while designing the vector to prevent recombination between the wild-type HIV-1. During acute infection, HIV RNA is detectable in 0.01-1% of peripheral T cells, compared with 60% of gastrointestinal tract mucosal memory CD4+ T cells (Costiniuk and Angel, 2012) thus, to start the vector replication the gut-associated lymphoid tissue would be good place of vector injection and minimize the amount of vector needed for start.

Having vector able to spread systematically, which mimic the HIV, gives in this example a big advantage to the previously used methods. This vector would not be eradicated by the immune system as it would escape the human antibodies in the same way as the wild-type HIV. The vector also would be able to replicates in HIV-1 positive cell and perhaps, to reach all available cells without causing new latency. Similar methods in which a vector can be used against another virus may have in the future application against other viruses. However, there is need to find the right modification in the construct by series of experiments before the right vector could be obtained.

In addition, increased and unstoppable vector replication would lead to host cells apoptosis. The modified HIV vector, once packaged with all viral proteins gaining from the wild-type HIV would be able to maturates and infect other cells further. However, if the missing protein is not obtained during process of maturation from the wild-type virus which for instance could be not present during the vector transcription, that would stop production of vector particles able to maturates and capable to deliver it genetic material into another living cells. In other words this method showing that the vector must hijacks or "parasite" any missing HIV component, not present in the vector genome from the wild-type HIV expression and deprive the wild-type HIV from accessing this protein preventing the wild-type HIV from budding. In order to achieve this effect, the vector's transcription and production of unspliced RNA must be increased. The vector, once having suppresses the availability to obtain the missing component to the wild-type HIV, would terminate the wild-type HIV maturation, latency and spread. Lack of wild-type HIV producing protein necessary for vector replication competence would terminate vector replication. Increased apoptosis in all reservoirs cells infected by mutant and wild-type HIV would lead to total depletion of the HIV reservoir and provides a novel approach for treatment or prevention of HIV infection.

Non-patent citations:

Chin, Μ. P., Chen, J., Nikolaitchik, O. A., & Hu, W. S. (2007). Molecular determinants of HIV-1 intersubtype recombination potential. Virology, 363(2), 437-446.

Chomont, N., El-Far, M., Ancuta, P., Trautmann, L., Procopio, F. A., Yassine-Diab, B., Sekaly, R. P. (2009). HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nature medicine, 15(8), 893-900.

Costiniuk, C. T., Angel, J. B. (2012). Human immunodeficiency virus and the gastrointestinal immune system: does highly active antiretroviral therapy restore gut immunity&quest. Mucosal immunology, 5(6), 596-604.

Dirac AM, Huthoff H, Kjems J, Berkhout B: Requirements for RNA heterodimerization of the human immunodeficiency virus type 1 (HIV-1) and HIV-2 genomes. J. Gen. Virol. 83(Pt 10), 2533-2542 (2002).

Duverger, A., Wolschendorf, F., Zhang, M., Wagner, F., Hatcher, B., Jones, J., Kutsch, O. (2013). An AP-1 binding site in the enhancer/core element of the HIV-1 promoter controls the ability of HIV-1 to establish latent infection.Journal of virology, 87(4), 2264-2277.

Finzi, D., Blankson, J., Siliciano, J. D., Margolick, J. B., Chadwick, K., Pierson, T., Siliciano, R. F. (1999). Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nature medicine, 5(5), 512-517.

Kim, K. C., Kim, H. G., Roh, T. Y., Park, J., Jung, K. M., Lee, J. S., Choi, B. S. (2011). The effect of CD4 receptor downregulation and its downstream signaling molecules on HIV-1 latency. Biochemical and biophysical research communications, 404(2), 646-651.

Nikolaitchik, O. A., Dilley, K. A., Fu, W., Gorelick, R. J., Tai, S. H. S., Soheilian, F., Flu, W. S. (2013). Dimeric RNA recognition regulates HIV-1 genome packaging. PLoS pathogens, 9(3), el003249.

Patel, P., Ansari, Μ. Y., Bapat, S., Thakar, M., Gangakhedkar, R., Jameel, S. (2014). The microRNA miR-29a is associated with human immunodeficiency virus latency. Retrovirology, 11(1), 108-108.

Perelson, A. S., Neumann, A. U., Markowitz, M., Leonard, J. M., Flo, D. D. (1996). HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science, 271(5255), 1582-1586.

Suzuki, K., Hattori, S., Marks, K., Ahlenstiel, C., Maeda, Y., Ishida, T., Kelleher, A. D. (2013). Promoter targeting shRNA suppresses HIV-1 infection in vivo through transcriptional gene silencing. Molecular Therapy—Nucleic Acids, 2(12), el37.

Claims (15)

CLAIMS:

1. A modified human immunodeficiency virus (HIV) genome vector, characterized in the vector to be a replication-incompetent in a living cell or organism which is free from the HIV virus during the time of presence of the vector genome, but replication-competent in the presence of the wild-type HIV virus in the same living cell or organism, due to competition in obtaining one or more HIV building protein from the wild-type or mutant HIV, needed during the process of HIV assembly or maturation to gain infectivity by the vector.

2. The method of claim 1, wherein the vector is used to prevent or terminate infection caused by any genotypic variants or mutants of HIV in HIV-positive or HIV-negative patients.

3. The method of claim 1 and 2, wherein the modification of the HIV to obtain a vector replication-incompetent in HIV free cell and replication-competent in HIV positive cell, results in modification of HIV genetic sequences of at least one or more nucleotides in at least one or more following sequences: Reverse Transcriptase, Integrase, Protease, Matrix Protein, Capsid Protein, Viral Infectivity Factor, gpl20, gp41, Nucleocapsid Protein, Viral Protein R, P6 Protein, Negative Regulatory Factor, Regulatory Of Virion Protein, Transactivation of Transcription Proteins, nucleotide sequences from RNA or DNA originated from any genotypic variants or mutants of HIV or host cell genome which binds directly to the HIV viral RNA sequences, or RNA originated form HIV during process of HIV assembly or maturation or sequences which are part of DNA or RNA binding complexes associated during HIV assembly or maturation.

4. The method of claim 3, wherein the alteration in vector sequences applies to deletion or insertion or exchange or chemical modification of at least one or more nucleotides presented in the wild-type HIV or any genotypic variants or mutants of HIV.

5. The method of claim 1, wherein the vector sequence is packaged and delivered into the cells using any genotypic variants of HIV, or via non-viral vector method.

6. The method of claim 1, wherein the phrase "wild-type HIV" applies to any genotypic variants or mutants of HIV and any subtype of viruses belonging to genus Lentivirus.

7. The method of claim 1, 2 and 6, wherein the viral vector is administrated on, from one to seven days weekly, to the living organism which have or have not antibody for viral genetic material including the viral building block proteins.

8. The method of claim 3, in which the eradication of virus comprises one or more additional active agents.

9. The method of claim 8, in which the one or more additional active agent is one or more of antiviral agents, an antibiotic, an antiemetic, a cell-based immunotherapeutic, a protein-based therapeutic, a vector comprising a gene for producing a therapeutic or immunotherapeutic composition, an cytotoxic agent, a radio-immunotherapy or virus replication inhibitor.

10. The method of claim 1, 2 and 3, wherein the viral vector genetic sequence can integrate into living cell genome or stabilized floating in the cytoplasm or nucleus.

11. The method of claim 10, wherein the vector can obtain missing viral proteins necessary to gain replication-competence from the wild-type HIV if the wild-type HIV is also present inside the same infected cell.

12. The method of claim 3 and 11, wherein the obtaining of altered, deleted or modified protein from the wild-type HIV allows transformation of the vector into replication-competent virus able to leave the cells and further infect other living cells.

13. The method of claim 2, wherein the eradication of the HIV virus is due to vector replication combined with the host cells apoptosis and suppression of maturation of the HIV due to the lack of accessibility of the protein produced by the wild-type HIV but utilization by the vector as a result of increased transcription compared with wild-type HIV.

14. The method of claim 1, wherein the process of eradicating of viral infection due to competition for one or more viral protein between vector and the wild-type virus can be applied to eradicate viral infections from virus family belong to any of the following: Adenoviridae, Papovaviridae, Parvoviridae, Herpesviridae, Poxviridae, Hepadnaviridae, Anelloviridae. Reoviridae. Picornaviridae. Caliciviridae. Togaviridae. Arenaviridae. Flaviviridae. Orthomvxoviridae. Paramvxoviridae. Bunvaviridae. Rhabdoviridae. Filoviridae. Coronaviridae. Astroviridae. Bornaviridae. Arteriviridae. Hepeviridae, Retroviridae and apply to any protein sequences presented in these families.

15. The method of claim 1 and 14, wherein the vector replication is increased, lowered or unchanged compared with the wild-type virus due to genetic engineering of one or more viral genomic sequences which are responsible for controlling the virus concentration level.