RNAi can be introduced into cells by either the "expressed" or the "delivered" route, depending on whether the RNAi molecules are expressed in cells or whether they are delivered exogenously. Benitec´s patented DNA-directed RNA interference (ddRNAi) technology uses the "expressed" pathway. It involves inserting a DNA construct into a cell, triggering the production of double stranded RNA (dsRNA) that is immediately cleaved into small interfering RNA (siRNA), which then enter the cellular RNAi pathway and causes the destruction of the rogue gene or viral genomes.
DNA-directed RNAi or ddRNAi is used to produce a dsRNA inside the cell. By introducing a DNA construct into a cell, Benitec's ddRNAi technology triggers the production of double stranded RNA (dsRNA), which is then cleaved into small interfering RNA (siRNA) by Dicer, a specific type of RNAse III, as part of the RNAi process. Consequently both strategies operate using a similar mechanism however both applications have strengths and weaknesses and thus different therapeutic applications.
Scientists at Benitec Limited in collaboration with the City of Hope are investigating a method to fight HIV, the virus that causes AIDS, with genetically modified stem cells. But that is not all. At the same time it seeks to knock down virus levels, the technique aims to eradicate the lymphoma that arises in many HIV-positive patients. The treatment is the first to use specially engineered, HIV-fighting genes that are inserted into patients´ own harvested stem cells. The cells would then be reinfused into HIV-infected patients during a bone marrow transplant. If successful, the new treatment could allow patients´ bodies to produce HIV-resistant white blood cells indefinitely. Scientists use a lentivirus to carry the gene segments into the stem cells. The lentivirus vector encodes 3 forms of anti-HIV RNA: RNAi in the form of a short hairpin RNA (shRNA) targeted to an exon in HIV-1 tat/rev (shI), a decoy for the HIV TAT-reactive element (TAR), and a ribozyme that targets the host cell CCR5 chemokine receptor (CCR5RZ).
This new trial is the first time that scientists will use three different anti- HIV segments at one time, which they hope will prevent HIV from developing resistance. The virus can mutfate around any one element, but it´s hard to mutate around three things. This study with City of Hope is entitled, "A pilot study of the safety and feasibility of stem cell therapy for AIDS lymphoma using stem cells treated with a lentiviral vector-encoding multiple anti-HIV RNA´s." This is Benitec´s first human trial and will involve HIV-1 infected adults 18-60 years old who suffer AIDS - related lymphoma. A primary objective of this study is to determine the safety and feasibility of Benitec´s RNAi technology in treating these patients. The study has FDA approval to commence with patients being recruited and screened at City of Hope and should be completed in approximately 12 months.
Benitec is also in collaboration with the City of Hope on a T cell approach using the same vector. Benitec has also licensed its Hepatitis C programme to Tacere Therapeutics Inc for further development.
Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), has infected over 40 million people worldwide and caused 3 million deaths in 2003. AIDS-related lymphoma is the occurrence of lymphatic cancer in AIDS patients. AIDS-related lymphoma usually grows and spreads faster to other parts of the body more often than lymphoma that is not related to AIDS. About 10% of HIV infections lead to AIDS-related lymphoma (about 4000 patients in the USA).
Although there is no cure for HIV/AIDS, there are a number of medical treatments available to help keep HIV in check and prevent progression to AIDS. Highly Active Anti Retroviral Therapy (HAART) is the standard of care for HIV infection. HAART has made AIDS more manageable, although it has severe side effects and patients remain infectious. Furthermore, the high mutation rate of HIV makes multiple drug resistance a continuing and challenging problem.
Treatment of AIDS lymphoma is extremely difficult. Autologous bone marrow stem cell (BMSC) transplantation after chemotherapy is the only therapy currently available, but HIV rapidly re-establishes in the newly derived blood cells, helped by the fact that HAART is temporarily stopped during the procedure.
Kim, D.H. and Rossi, J.J. (2007) Strategies for silencing human disease using RNA interference. Nat. Rev. Genet. 8: 173-184. Since the first description of RNA interference (RNAi) in animals less than a decade ago, there has been rapid progress towards its use as a therapeutic modality against human diseases. Advances in our understanding of the mechanisms of RNAi and studies of RNAi in vivo indicate that RNAi-based therapies might soon provide a powerful new arsenal against pathogens and diseases for which treatment options are currently limited. Recent findings have highlighted both promise and challenges in using RNAi for therapeutic applications. Design and delivery strategies for RNAi effector molecules must be carefully considered to address safety concerns and to ensure effective, successful treatment of human diseases.
Morris, K.V. and Rossi, J.J. (2006) Antiviral applications of RNAi. Curr. Opin. Mol. Ther. 8: 115-121. RNA interference (RNAi) is a natural mechanism by which small interfering RNA (siRNA) operates to specifically and potently downregulate the expression of a target gene. This downregulation has been thought to predominantly function at the level of mRNA, as post-transcriptional gene silencing. The discovery that siRNAs can suppress gene expression at the level of transcription, that is, transcriptional gene silencing, has created a major paradigm shift in mammalian RNAi. These findings significantly broaden the role that RNA, specifically siRNA and potentially microRNA, plays in the regulation of gene expression, as well as the breadth of potential siRNA target sites. Indeed, the specificity and simplicity of design makes the use of siRNAs to target and suppress virtually any gene of interest a realized technology. Furthermore, since siRNAs are small nucleic acid reagents, they are unlikely to elicit an immune response, theoretically making them good therapeutics. The development, delivery and potential therapeutic use of antiviral siRNAs in treating viral infections and emerging viral threats are reviewed.
Morris, K.V. and Rossi, J.J. (2006) Lentivirus-mediated RNA interference therapy for human immunodeficiency virus type 1 infection. Hum. Gene Ther. 17: 479-486. RNA interference (RNAi) is a natural mechanism by which small interfering RNAs (siRNAs) operate to specifically and potently downregulate the expression of a target gene. This downregulation has been demonstrated by targeting siRNAs to the mRNA (posttranscriptional gene silencing) as well as to the gene promoter, regulating gene expression epigenetically by transcriptional gene silencing. These observations significantly broaden the role RNA plays in the cell and suggest that siRNAs could prove to be a potent future therapeutic for the treatment of diseases such as human immunodeficiency virus type 1 (HIV-1) infection. The specificity and simplicity of design and the ability to express siRNAs from mammalian promoters make the use of siRNAs to target and suppress virtually any gene or gene promoter of interest a soon-to-be-realized technology. However, the delivery and stable expression of siRNAs to target cells remain an enigma that could be surmounted, at least regarding the treatment of HIV-1 infection, by the application of lentiviral vectors to deliver and express anti-HIV-1 siRNAs in target cells. This review focuses on the development, delivery, and potential therapeutic use of antiviral siRNAs in treating HIV-1.
Unwalla, H.J., Li, H.T., Bahner, I., Li, M.J., Kohn, D. and Rossi, J.J. (2006) Novel Pol II fusion promoter directs human immunodeficiency virus type 1-inducible coexpression of a short hairpin RNA and protein. J. Virol. 80: 1863-1873. We demonstrate a novel approach for coexpression of a short hairpin RNA (shRNA) with an open reading frame which exploits transcriptional read-through of a minimal polyadenylation signal from a Pol II promoter. We first observed efficient inducible expression of enhanced green fluorescent protein along with an anti-rev shRNA. We took advantage of this observation to test coexpression of the transdominant negative mutant (humanized) of human immunodeficiency type 1 (HIV-1) Rev (huRevM10) along with an anti-rev shRNA via an HIV-1-inducible fusion promoter. The coexpression of the shRNA and transdominant protein resulted in potent, long-term inhibition of HIV-1 gene expression and suppression of shRNA-resistant mutants. This dual expression system has broad-based potential for other shRNA applications, such as cases where simultaneous knockdown of mutant and wild-type transcripts must be accompanied by replacement of the wild-type protein.
Snove, O., Jr. and Rossi, J.J. (2006) Expressing short hairpin RNAs in vivo. Nature Methods 3: 689-695. Promoter-based expression of short hairpin RNAs (shRNAs) may in principle provide stable silencing of genes in any tissue. As for all approaches that require transgene expression, safe delivery is the biggest obstacle, but toxicity can also occur via expression of the sequence itself. Innate immunity mechanisms can be triggered by expressed hairpin RNAs, critical cellular factors can be saturated, and genes other than the intended target can be silenced. Nevertheless, shRNAs constitute a valuable tool for in vivo research and have great therapeutic potential if the challenges with delivery and side effects are appropriately addressed.
Robbins, M.A., Li, M., Leung, I., Li, H., Boyer, D.V., Song, Y., Behlke, M.A. and Rossi, J.J. (2006) Stable expression of shRNAs in human CD34+ progenitor cells can avoid induction of interferon responses to siRNAs in vitro. Nature Biotechnol. 24: 566-571. RNA interference occurs when cytoplasmic small interfering RNAs (siRNAs) enter the RNA-induced silencing complex and one strand guides cleavage of the target RNA by the Argonaute 2 protein. A significant concern when applying siRNAs or expressing small hairpin RNAs (shRNAs) in human cells is activation of the interferon (IFN) response. Synthetic siRNAs harboring certain motifs can induce an immune response when delivered to mouse and human immune cells such as peripheral blood mononuclear cells, monocytes, plasmacytoid dendritic cells (pDCs) and nonplasmacytoid dendritic cells (mDCs). In the present study we have tested the immunostimulatory effects of lipid-delivered siRNAs versus Pol III promoter-expressed shRNAs in primary CD34+ progenitor-derived hematopoietic cells. We show that in this system, lipid-delivered siRNAs are potent inducers of IFNalpha and type I IFN gene expression, whereas the same sequences when expressed endogenously are nonimmunostimulatory.
Li, M., Li, H. and Rossi, J.J. (2006) RNAi in combination with a ribozyme and TAR decoy for treatment of HIV infection in hematopoietic cell gene therapy. Ann. N.Y. Acad. Sci. 1082: 172-179. Combinatorial therapies for the treatment of HIV infection have changed the course of the AIDS epidemic in developed nations where the antiviral drug combinations are readily available. Despite this progress, there are many problems associated with chemotherapy for AIDS including toxicities and emergence of viral mutants resistant to the drugs. Our goal has been the development of a hematopoietic gene therapy treatment for HIV infection. Like chemotherapy, gene therapy for treatment of HIV infection should be used combinatorially. We have thus combined three different inhibitory genes for treatment of HIV infection into a single lentiviral vector backbone. The inhibitory agents engage RNAi via a short hairpin RNA targeting HIV tat/rev mRNAs, a nucleolar localizing decoy that binds and sequesters the HIV Tat protein, and a ribozyme that cleaves and downregulates the CCR5 chemokine receptor used by HIV for cellular entry. This triple combination has proven to be highly effective for inhibiting HIV replication in primary hematopoietic cells, and is currently on track for human clinical application.
Li, M.-J., Kim, J., Li, S., Zaia, J., Yee, J.-K., Anderson, J., Akkina, R. and Rossi, J.J. (2005) Long-term inhibition of HIV-1 infection in primary hematopoietic cells by lentiviral vector delivery of a triple combination of anti-HIV shRNA, anti-CCR5 ribozyme, and a nucleolar-localizing TAR decoy. Mol. Ther. 12: 900-909. Combinatorial therapies for the treatment of HIV-1 infection have proven to be effective in reducing patient viral loads and slowing the progression to AIDS. We have developed a series of RNA-based inhibitors for use in a gene therapy-based treatment for HIV-1 infection. The transcriptional units have been inserted into the backbone of a replication-defective lentiviral vector capable of transducing a wide array of cell types, including CD34+ hematopoietic progenitor cells. The combinatorial therapeutic RNA vector harbors a U6 Pol III promoter-driven short hairpin RNA (shRNA) targeting the rev and tat mRNAs of HIV-1, a U6 transcribed nucleolar-localizing TAR RNA decoy, and a VA1-derived Pol III cassette that expresses an anti-CCR5 ribozyme. Each of these therapeutic RNAs targets a different gene product and blocks HIV infection by a distinct mechanism. Our results demonstrate that the combinatorial vector suppresses HIV replication long term in a more-than-additive fashion relative to the single shRNA or double shRNA/ribozyme or decoy combinations. Our data demonstrate the validity and efficacy of a combinatorial RNA-based gene therapy for the treatment of HIV-1 infection.
Unwalla, H.J., Li, M.J., Kim, J.D., Li, H.T., Ehsani, A., Alluin, J. and Rossi, J.J. (2004) Negative feedback inhibition of HIV-1 by TAT-inducible expression of siRNA. Nature Biotechnol 22: 1573-1578. Here we demonstrate that an inducible anti-HIV short hairpin RNA (shRNA) expressed from a Pol II promoter inhibits HIV-1 gene expression in mammalian cells. Our strategy is based on a promoter system in which the HIV-1 LTR is fused to the Drosophila hsp70 minimal heat shock promoter. This system is inducible by HIV-1 TAT, which functions in a negative feedback loop to activate transcription of an shRNA directed against HIV-1 rev. Upon induction the shRNA is processed to an siRNA that guides inhibition of HIV replication in cultured T-lymphocytes and hematopoietic stem cell-derived monocytes. The fusion promoter system may be safer than drug-inducible systems for shRNA-mediated gene therapy against HIV as the shRNAs are only expressed following HIV infection.
Banerjea A, Li MJ, Bauer G, Remling L, Lee NS, Rossi J and R Akkina (2003) Inhibition of HIV-1 by lentiviral vector-transduced siRNAs in T lymphocytes differentiated in SCID-hu mice and CD34+ progenitor cell-derived macrophages. Mol Ther 8: 62-71.
Lee, N.S., Dohjima, T., Bauer, G., Li, H., Li, M.-J., Ehsani, A., Salvaterra, P. and Rossi, J. (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnology 20(5): 500-505.
Li, M. J., Bauer, G., Michienzi, A., Yee, J. K., Lee, N. S., Kim, J., Li, S., Castanotto, D., Zaia, J., and Rossi, J. J. (2003) Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs. Mol Ther 8: 196-206.
Michienzi, A., Castanotto, D., Lee, N., Li, S., Zaia, J. A., and Rossi, J. J. (2003) RNA-mediated inhibition of HIV in a gene therapy setting. Ann N Y Acad Sci 1002: 63-71.
Scherer, L. J., and Rossi, J. J. (2003) Approaches for the sequence-specific knockdown of mRNA. Nat Biotechnol 21: 1457-14652
