#7 - Versatile Delivery Method for Cancer Therapeutics
Title: Virus-Like Particles as a Platform for Efficient Delivery of Proteins and RNA
NIH Reference No.: E-264-2011 and E-010-2008
Executive Summary:
General Description
Current methods of delivering proteins or RNA to mammalian cells are limited by a lack of target specificity and toxicity, among other shortcomings. Protein transduction is an emerging technology for delivering proteins into cells by exploiting the ability of certain proteins to penetrate the cell membrane. However, the majority of the proteins delivered by this means are usually trapped and subsequently degraded in the endosomes-lysosomes of recipient cells. Virus mediated gene delivery (gene therapy) have been widely implemented as a delivery method for the past two decades. Since viruses have unique ability infect cells and deliver the contents in the cytoplasm with almost 100% efficiency, two novel technologies were developed by NCI inventors to deliver proteins and RNAs, respectively, based on virus-like particles.
The first VLP technology is based on GAG structural protein of Rous sarcoma leukosis virus fused to protein of interest and a fusogenic envelope glycoprotein. These VLPs are devoid of retrovirus genomic RNA and structural proteins as: RT/POL and protease. Such particles are extremely efficient in delivering proteins to cells and the absence of mentioned RT/POL protein and retroviral genomic RNA in VLPs assembly make these particles safe and non-integrating platform for therapeutic use.
A second VLP technology utilized alphavirus replicon system. In this technology, VLPs were generated using alphavirus replicon coding sequences (+ RNA coding for alphavirus RNA dependent RNA polymerase followed by coding sequence of gene of interest), a Gag protein from Rous sarcoma leukosis virus and a fusogenic envelope protein. This new approach allows for safe incorporation of RNA(s) into VLPs formed by Gag and converts these VLPs into a very efficient reagent for RNA delivery and expression in target cells.
Virus-like particles (VLPs) have uses in immunization against microbial pathogens but also in immunotherapy and gene therapy for chronic diseases such as hypertension, Alzheimer’s disease, and cancer. VLPs assembled with foreign fragments on their surfaces could efficiently deliver materials to the designated targets, making them promising candidates for novel delivery system of RNA or proteins.
Scientific Progress
NCI inventors have developed novel VLPs that are capable of binding to and delivering materials within a target mammalian cell, including human cells. The VLPs are safer than viral delivery because they are incapable of re-infecting target cells. The VLPs can optionally comprise inhibitory recombinant polynucleotides, such as microRNA (miRNA), antisense RNA or small hairpin RNA, to down regulate or turn off expression of a particular gene within the target cell. One of the main drawbacks of current miRNA technologies is the delivery system and the low amounts of miRNA that are actually delivered to the target cells. This technology solves both of these problems as the miRNA encoded VLPs can facilitate microRNA production at a rate of approximately 106 copies per target cell. The inventors have demonstrated robust protein expression and miRNA mediated inhibition of gene expression. In addition, they have shown that immune response provoking proteins can be expressed in target cells and have done animal studies in mice using the 4T1 mouse mammary carcinoma cell line.
In addition to the miRNA containing VLPs, the VLPs can also deliver proteins into the target cell. The inventors have validated the biological activity of Cre recombinase and cytotoxic enzymes delivered using VLPs in a human prostate cancer cell line. Specific ligands such as TNF-related apoptosis-inducing ligand (TRAIL) can be displayed on the surface of hybrid VLPs. In vitro assays indicated that VLP-mediated TRAIL delivery is at least twice as effective and required 1000 times less peptide than soluble TRAIL, which is currently under clinical trial elsewhere.
Alternatively, recombinant polynucleotides packaged within VLPs can comprise a gene encoding a therapeutic protein so as to enable expression of that protein within the target cell. Specifically, VLPs of the invention are composed of an alphavirus replicon that contains a recombinant polynucleotide, a retroviral gag protein, and a fusogenic envelope glycoprotein. While the claimed VLPs have a variety of applications, therapeutic uses of the VLPs include directing antibody synthesis and converting cancer cells into antigen presenting cells.
Strengths
Weaknesses
Patent Status
For E-264-2011: PCT application No. PCT/US13/31876 filed 15 March 2013 (Unpublished)
For E-010-2008: U.S. Patent Application No. 13/122,513 filed 04 April 2011 Google Patent
Relevant Publications
Kaczmarczyk SJ, Sitaraman K, Young HA, Hughes SH, Chatterjee DK. PNAS, 2011 108(41): 16998–17003
(PMID: 21949376)
Inventor Bios
Stanislaw J Kaczmarczyk, Ph.D.
Dr. Kaczamrczyk received his BSC from La Trobe University, Master of Biotechnology from Monash University and his Ph.D. degrees from University of Melbourne, Australia. He acquired a sound knowledge of molecular biology techniques during master of technology training and this allowed him to propose and successfully complete his Ph.D. project encompassing the etiology of Diabetes Type II using mouse models of conditional spatio-temporal gene inactivation mediated through novel Cre-Lox technology.
His postdoctoral work at NIH involved further expansion of Cre-lox technology and resulted in successful design of numerous inducible gene switch technologies which were implemented in transgenic animal models of cancer. The postdoctoral training at NCI in Retroviral Replication and Vector Design Lab under mentorship of Dr. S. Hughes allowed him to implement gained knowledge to develop such technologies as chimeric virus like particles (VLPs) carrying RNA for alpha virus replicon. This later technology is currently used for cancer immunotherapy of mammary tumor in mouse model. He retains authorship on number of peer reviewed scientific publications and patents applications.
Deb Chatterjee, Ph.D.
Dr. Chatterjee is the Associate Director of the Protein Expression Laboratory within the Advanced Technology Program in the Frederick National Laboratory for Cancer Research through SAIC-Frederick, Inc. Dr. Chatterjee received his B.Sc (Chemistry Hons). and M.Sc. (Biochemistry) degrees from the University of Calcutta, India. His postdoctoral work, in the laboratory of Prof. A.M. Chakrabarty at the University of Illinois at Chicago, involved genetic engineering Pseudomonas and Caulobacter strains for their ability to degrade toxic aromatic compounds including chlorobenzoates, Agent Orange, toluene, and benzoates.
Following post-doctoral research, Dr. Chatterjee joined at Life Technologies in Gaithersburg, Maryland. He held positions from senior scientist to Director of R&D of Life Technologies. He initiated and headed a group for cloning, engineering and expression of many biotechnologically important proteins and enzymes, such as restriction enzymes, reverse transcriptases (from MMLV, RSV, and AMV) and DNA polymerases (from E. coli, T7 and T5 phages, and thermostable organisms). Products developed from his innovation generate hundreds of millions dollars in revenue for Life technologies every year. He invented a mutation that allows DNA polymerases to efficiently incorporate di-deoxy and fluorescently labeled nucleotides into DNA. An estimated 99 percent of the genomic sequences in databases were determined with enzymes containing this mutation.
In 2002, Dr. Chatterjee joined the National Cancer Institute, SAIC-Frederick, Inc as a leader to develop novel and innovative technologies for cancer research and therapy. His group has developed a number novel technologies: (a) a cell-free protein synthesis system that produces proteins more than 10-folds compared to any commercially available sources, (b) a novel on-demand protein microarray system that converts a DNA array into protein array on demand----the DNA in the array makes the proteins in situ and captures the proteins as well, (c) a novel protein delivery system using virus-like particles that could be used for delivering protein drugs to specific target cells and (d) VLP mediated RNA delivery. His group is also trying to develop other technologies—generation of calibrator protein kinases for assessing kinase inhibitors during clinical studies, cancer immunotherapy, etc. Dr. Chatterjee has published 56 articles and holds 37 U.S. patents.
NIH Reference No.: E-264-2011 and E-010-2008
Executive Summary:
- Category: Vaccine or Drug Delivery (Protein or RNA)
- Disease Focus: Cancer broadly
- Basis of Invention: Virus-like Particle (VLP)
- How it works: A platform technology for delivery of proteins or RNA to cancer cells
- Patent Status: PCT Application filed; U.S. Patent Pending
- Lead Inventors: Stanislaw J Kaczmarczyk, Ph.D. and Deb Chatterjee, Ph.D.
- Development Stage: Preclinical, in vitro data available for both miRNA and protein delivery; verified robust protein delivery, robust protein expression, miRNA mediated inhibition of gene expression and expression of innate immune response provoking proteins using coding RNA in target cells in vitro. Pre-clinical in-vitro studies for protein delivery with human prostate cancer cell lines. Mouse in vivo data using 4T1 mouse mammary carcinoma cell line as a model system for cancer immunotherapy
- Novelty: Less toxic and more target specificity than current delivery methods; eliminates the need for expensive antigen purification methods
- Clinical Application: Delivery of proteins or miRNA or coding RNA into cells, direct antibody production by in vivo injection of replicons, in vivo cancer treatment by converting cancer cells into antigen presenting cells.
General Description
Current methods of delivering proteins or RNA to mammalian cells are limited by a lack of target specificity and toxicity, among other shortcomings. Protein transduction is an emerging technology for delivering proteins into cells by exploiting the ability of certain proteins to penetrate the cell membrane. However, the majority of the proteins delivered by this means are usually trapped and subsequently degraded in the endosomes-lysosomes of recipient cells. Virus mediated gene delivery (gene therapy) have been widely implemented as a delivery method for the past two decades. Since viruses have unique ability infect cells and deliver the contents in the cytoplasm with almost 100% efficiency, two novel technologies were developed by NCI inventors to deliver proteins and RNAs, respectively, based on virus-like particles.
The first VLP technology is based on GAG structural protein of Rous sarcoma leukosis virus fused to protein of interest and a fusogenic envelope glycoprotein. These VLPs are devoid of retrovirus genomic RNA and structural proteins as: RT/POL and protease. Such particles are extremely efficient in delivering proteins to cells and the absence of mentioned RT/POL protein and retroviral genomic RNA in VLPs assembly make these particles safe and non-integrating platform for therapeutic use.
A second VLP technology utilized alphavirus replicon system. In this technology, VLPs were generated using alphavirus replicon coding sequences (+ RNA coding for alphavirus RNA dependent RNA polymerase followed by coding sequence of gene of interest), a Gag protein from Rous sarcoma leukosis virus and a fusogenic envelope protein. This new approach allows for safe incorporation of RNA(s) into VLPs formed by Gag and converts these VLPs into a very efficient reagent for RNA delivery and expression in target cells.
Virus-like particles (VLPs) have uses in immunization against microbial pathogens but also in immunotherapy and gene therapy for chronic diseases such as hypertension, Alzheimer’s disease, and cancer. VLPs assembled with foreign fragments on their surfaces could efficiently deliver materials to the designated targets, making them promising candidates for novel delivery system of RNA or proteins.
Scientific Progress
NCI inventors have developed novel VLPs that are capable of binding to and delivering materials within a target mammalian cell, including human cells. The VLPs are safer than viral delivery because they are incapable of re-infecting target cells. The VLPs can optionally comprise inhibitory recombinant polynucleotides, such as microRNA (miRNA), antisense RNA or small hairpin RNA, to down regulate or turn off expression of a particular gene within the target cell. One of the main drawbacks of current miRNA technologies is the delivery system and the low amounts of miRNA that are actually delivered to the target cells. This technology solves both of these problems as the miRNA encoded VLPs can facilitate microRNA production at a rate of approximately 106 copies per target cell. The inventors have demonstrated robust protein expression and miRNA mediated inhibition of gene expression. In addition, they have shown that immune response provoking proteins can be expressed in target cells and have done animal studies in mice using the 4T1 mouse mammary carcinoma cell line.
In addition to the miRNA containing VLPs, the VLPs can also deliver proteins into the target cell. The inventors have validated the biological activity of Cre recombinase and cytotoxic enzymes delivered using VLPs in a human prostate cancer cell line. Specific ligands such as TNF-related apoptosis-inducing ligand (TRAIL) can be displayed on the surface of hybrid VLPs. In vitro assays indicated that VLP-mediated TRAIL delivery is at least twice as effective and required 1000 times less peptide than soluble TRAIL, which is currently under clinical trial elsewhere.
Alternatively, recombinant polynucleotides packaged within VLPs can comprise a gene encoding a therapeutic protein so as to enable expression of that protein within the target cell. Specifically, VLPs of the invention are composed of an alphavirus replicon that contains a recombinant polynucleotide, a retroviral gag protein, and a fusogenic envelope glycoprotein. While the claimed VLPs have a variety of applications, therapeutic uses of the VLPs include directing antibody synthesis and converting cancer cells into antigen presenting cells.
Strengths
- Robust expression of miRNA at target cells with approximately 106 copies per cell
- Fast expression (approx. 3-4 hrs compared to 1-2 days) following VLP introduction into target cells
Weaknesses
- Synthesized VLPs need to be characterized for structural integrity, stability, and components, including the encapsidated nucleic acids, which require advanced EM techniques and sophisticated analysis.
- The VLP technology still needs to be validated in larger animal models
Patent Status
For E-264-2011: PCT application No. PCT/US13/31876 filed 15 March 2013 (Unpublished)
For E-010-2008: U.S. Patent Application No. 13/122,513 filed 04 April 2011 Google Patent
Relevant Publications
Kaczmarczyk SJ, Sitaraman K, Young HA, Hughes SH, Chatterjee DK. PNAS, 2011 108(41): 16998–17003
(PMID: 21949376)
Inventor Bios
Stanislaw J Kaczmarczyk, Ph.D.
Dr. Kaczamrczyk received his BSC from La Trobe University, Master of Biotechnology from Monash University and his Ph.D. degrees from University of Melbourne, Australia. He acquired a sound knowledge of molecular biology techniques during master of technology training and this allowed him to propose and successfully complete his Ph.D. project encompassing the etiology of Diabetes Type II using mouse models of conditional spatio-temporal gene inactivation mediated through novel Cre-Lox technology.
His postdoctoral work at NIH involved further expansion of Cre-lox technology and resulted in successful design of numerous inducible gene switch technologies which were implemented in transgenic animal models of cancer. The postdoctoral training at NCI in Retroviral Replication and Vector Design Lab under mentorship of Dr. S. Hughes allowed him to implement gained knowledge to develop such technologies as chimeric virus like particles (VLPs) carrying RNA for alpha virus replicon. This later technology is currently used for cancer immunotherapy of mammary tumor in mouse model. He retains authorship on number of peer reviewed scientific publications and patents applications.
Deb Chatterjee, Ph.D.
Dr. Chatterjee is the Associate Director of the Protein Expression Laboratory within the Advanced Technology Program in the Frederick National Laboratory for Cancer Research through SAIC-Frederick, Inc. Dr. Chatterjee received his B.Sc (Chemistry Hons). and M.Sc. (Biochemistry) degrees from the University of Calcutta, India. His postdoctoral work, in the laboratory of Prof. A.M. Chakrabarty at the University of Illinois at Chicago, involved genetic engineering Pseudomonas and Caulobacter strains for their ability to degrade toxic aromatic compounds including chlorobenzoates, Agent Orange, toluene, and benzoates.
Following post-doctoral research, Dr. Chatterjee joined at Life Technologies in Gaithersburg, Maryland. He held positions from senior scientist to Director of R&D of Life Technologies. He initiated and headed a group for cloning, engineering and expression of many biotechnologically important proteins and enzymes, such as restriction enzymes, reverse transcriptases (from MMLV, RSV, and AMV) and DNA polymerases (from E. coli, T7 and T5 phages, and thermostable organisms). Products developed from his innovation generate hundreds of millions dollars in revenue for Life technologies every year. He invented a mutation that allows DNA polymerases to efficiently incorporate di-deoxy and fluorescently labeled nucleotides into DNA. An estimated 99 percent of the genomic sequences in databases were determined with enzymes containing this mutation.
In 2002, Dr. Chatterjee joined the National Cancer Institute, SAIC-Frederick, Inc as a leader to develop novel and innovative technologies for cancer research and therapy. His group has developed a number novel technologies: (a) a cell-free protein synthesis system that produces proteins more than 10-folds compared to any commercially available sources, (b) a novel on-demand protein microarray system that converts a DNA array into protein array on demand----the DNA in the array makes the proteins in situ and captures the proteins as well, (c) a novel protein delivery system using virus-like particles that could be used for delivering protein drugs to specific target cells and (d) VLP mediated RNA delivery. His group is also trying to develop other technologies—generation of calibrator protein kinases for assessing kinase inhibitors during clinical studies, cancer immunotherapy, etc. Dr. Chatterjee has published 56 articles and holds 37 U.S. patents.