Hypoxia-directed tumor targeting of CRISPR-Cas9 and HSV-TK suicide gene therapy using lipid nanoparticles

Abstract

Hypoxia is a characteristic feature of solid tumors that contributes to tumor aggressiveness and is associated with resistance to cancer therapy. The hypoxia inducible factor-1 (HIF-1) transcription factor complex mediates hypoxia-specific gene expression by binding to hypoxia-responsive element (HRE) sequences within the promoter of target genes. HRE-driven expression of therapeutic cargo has been widely explored as a strategy to achieve cancer-specific gene expression. By utilizing this system, we achieve hypoxia-specific expression of two therapeutically relevant cargo elements: the herpes simplex virus thymidine kinase (HSV-tk) suicide gene and the CRISPR-Cas9 nuclease. Using an expression vector containing five copies of the HRE derived from the vascular endothelial growth factor gene, we are able to show high transgene expression in cells in a hypoxic environment, similar to levels achieved using the cytomegalovirus (CMV) and CBh promoters. Furthermore, we are able to deliver our therapeutic cargo to tumor cells with high efficiency using plasmid-packaged lipid nanoparticles (LNPs) to achieve specific killing of tumor cells in hypoxic conditions while maintaining tight regulation with no significant changes to cell viability in normoxia. Hypoxia is a characteristic feature of solid tumors that contributes to tumor aggressiveness and is associated with resistance to cancer therapy. The hypoxia inducible factor-1 (HIF-1) transcription factor complex mediates hypoxia-specific gene expression by binding to hypoxia-responsive element (HRE) sequences within the promoter of target genes. HRE-driven expression of therapeutic cargo has been widely explored as a strategy to achieve cancer-specific gene expression. By utilizing this system, we achieve hypoxia-specific expression of two therapeutically relevant cargo elements: the herpes simplex virus thymidine kinase (HSV-tk) suicide gene and the CRISPR-Cas9 nuclease. Using an expression vector containing five copies of the HRE derived from the vascular endothelial growth factor gene, we are able to show high transgene expression in cells in a hypoxic environment, similar to levels achieved using the cytomegalovirus (CMV) and CBh promoters. Furthermore, we are able to deliver our therapeutic cargo to tumor cells with high efficiency using plasmid-packaged lipid nanoparticles (LNPs) to achieve specific killing of tumor cells in hypoxic conditions while maintaining tight regulation with no significant changes to cell viability in normoxia. IntroductionOver recent years, many advances have been made in cancer therapy, including the development of targeted inhibitors and immunotherapies with greatly reduced adverse effects. Despite this, the current 5-year survival rate among all cancers is only 68%, dropping to under 10% for certain cancer types, including pancreatic cancer and glioblastoma.1Howlader N, Noone AM, Krapcho M, Miller D, Brest A, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, et al. (eds). SEER Cancer Statistics Review, 1975-2018, National Cancer Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2018/, based on November 2020 SEER data submission, posted to the SEER web site, April 2021.Google Scholar The resistance of these high-risk cancers to current therapies begets a need to develop novel treatment modalities to improve patient survival and quality of life. Several unique cancer treatment strategies have emerged by repurposing proteins found in viruses and bacteria to promote cell death, including the human herpes simplex virus thymidine kinase (HSV-tk) suicide gene and the CRISPR-Cas9 nuclease. Although both of these proteins have shown promise as treatment options for various cancers, there is a growing need to direct tumor-specific expression of these proteins in order to minimize the risk of off-target toxicity and, in the case of Cas9, off-target mutations.The hypoxic microenvironment offers a unique opportunity to target these exogenous proteins to regions of the tumor where the most aggressive and treatment-resistant cancer cells often reside. Solid tumors account for approximately 90% of all human malignancies.2Hematopoietic Cancers.in: Mak T.W. Saunders M.E. Jett B.D. Primer to the Immune Response. Academic Cell, 2014: 553-585Crossref Google Scholar As a result of vascular abnormalities that lead to low intratumoral blood flow, up to 50%–60% of locally advanced solid tumors develop areas of low O2 (<10 mmHg) partial pressure compared with their surrounding tissues.3Vaupel P. Mayer A. Hypoxia in cancer: significance and impact on clinical outcome.Cancer Metastasis Rev. 2007; 26: 225-239Crossref PubMed Scopus (1637) Google Scholar, 4Bernauer C. Stella Man Y.K. Chisholm J.C. Lepicard E.Y. Robinson S.P. Shipley J.M. Hypoxia and its therapeutic possibilities in paediatric cancers.Br. J. Cancer. 2020; 124: 539-551Crossref PubMed Scopus (16) Google Scholar, 5Höckel M. Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects.J. Natl. Cancer Inst. 2001; 93: 266-276Crossref PubMed Scopus (2131) Google Scholar This hypoxic state has been associated with increased tumor aggressiveness and resistance to current therapies.6Dhani A. M. The clinical significance of hypoxia in human PubMed Scopus Google Scholar all the proteins by hypoxic and their are the most the expression of is with a of approximately as a result of J. M. of factor is by an the Natl. A. PubMed Scopus Google factor in PubMed Scopus Google Scholar to within the of target including vascular endothelial growth factor containing copies of HRE sequences have been shown to specific gene expression in This gene expression has been in including hypoxia-specific cells and P. M. et cells and in solid PubMed Scopus Google M. of and in hypoxic tumor Google Scholar hypoxia-specific gene expression has been explored for the suicide gene gene using thymidine kinase and in and in PubMed Scopus Google J.M. of a hypoxia-responsive vector for tumor-specific gene PubMed Scopus Google Scholar we to improve on the and to this, regulation has been to the CRISPR-Cas9 to achieve specific expression of the CRISPR-Cas9 a unique opportunity to for tumor survival for no inhibitors By using HRE-driven we to cancer cell in hypoxic of the for is the of of and CRISPR-Cas9 in has been with levels of using A. and with the of suicide for targeted cancer Rev. PubMed Scopus Google Scholar, M. J. P. P. J. gene with by for with a PubMed Scopus Google Scholar, M. P. E.Y. and its PubMed Scopus Google Scholar significant for of CRISPR-Cas9 from the of the and the to direct in specific cell for in advances and PubMed Scopus Google Scholar the of therapies based on both and to virus 2020; PubMed Scopus Google Scholar are an to The promise and of in for PubMed Scopus Google Scholar with of in A. A. et of nanoparticles for 2020; PubMed Scopus Google Scholar, J. J. M. et for in the 2020; Scopus Google Scholar, J. C. J. et of lipid nanoparticles and in PubMed Scopus Google Scholar nanoparticles in a and to and therapeutic including and M.E. nanoparticles for Rev. 2020; PubMed Scopus Google Scholar, J. A. for cancer PubMed Scopus Google Scholar, nanoparticles for targeted from to J. PubMed Scopus Google Scholar, of CRISPR-Cas9 for specific 2020; PubMed Scopus Google Scholar, A. J.M. of lipid nanoparticles for in and in of PubMed Scopus Google Scholar many are made in order to the of to tumor A. et using targeted lipid nanoparticles for cancer 2020; PubMed Scopus Google J. M. of CRISPR-Cas9 and for tumor-specific gene with 2020; Scopus Google Scholar are to deliver cargo to the and a of for off-target nanoparticles gene from to clinical PubMed Scopus Google et in of nanoparticles for endothelial cell gene Natl. A. PubMed Scopus Google Scholar on these we to direct expression of and to and off-target the we on the development of both suicide gene and CRISPR-Cas9 therapeutic that expression in order to minimize off-target this we these from a promoter in with five copies of the HRE derived from the human using both hypoxia and we have cancer cell using two gene and of a order to the of for hypoxia-specific gene we a in cells in the of the hypoxia is a of that from P. M. 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M.E. of tumor by a thymidine kinase PubMed Scopus Google Scholar on these we and from the promoter of both the in both and and cells and the of the hypoxia hypoxia there is no expression of from the HRE expression under hypoxia is the as the with reduced as with cells only reduced cell viability in the of and the hypoxia hypoxia order to the hypoxic of in these we we in cells treatment in the these data that HRE-driven expression of is and cell viability only hypoxic expression and of the two to direct expression of The cytomegalovirus (CMV) promoter the expression of as the for these The promoter of a promoter and five copies of the human of cells with the HRE-driven cells in the of hypoxia for to for for viability of the in cells in with hypoxia cells with hypoxic conditions for a of in for cells with the and in the of with for for and the are as and are of expression of is a for gene that to CRISPR-Cas9 offers a unique opportunity to cancer by for tumor cell there is no to deliver and the CRISPR-Cas9 in cancer this many have on unique that are found only in cancer as and M. 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J. of suicide gene expression by promoter and regulation for cancer PubMed Scopus Google to utilizing of gene expression on the and advances in and with the Although most to the within a tumor are and This has been shown to result in of advances in tumor for cancer PubMed Scopus Google Scholar nanoparticles a and in solid J. tumor and therapeutic of cancer Scopus Google Scholar a we to to the of the to to tumor has emerged to direct and in specific and cell by an of an to the of an using and of CRISPR-Cas9 in A. et using targeted lipid nanoparticles for cancer 2020; PubMed Scopus Google Scholar Furthermore, there are and that to promote within a tumor in order to the hypoxic these to promote tumor of our and for in we the of and therapeutic cargo to hypoxic tumor cells using the of our hypoxia-specific expression and of the CRISPR-Cas9 in tumor cells using has been The in of the most aggressive and treatment-resistant cells within a solid tumor and explored for cancer and and and cell cells as a from the of of The human cell and to the in a with The and in this for and cell for using the the cells in using the and and cell the human cells cells using the to the by the of using to the by the of in this found in of cells with of the P. 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J. hypoxia to Scopus Google Scholar in the The in a of hypoxia cells in a for the in the hypoxia treatment the hypoxia cells with cells hypoxia for for hypoxia for and the result in a of in hypoxia using two the suicide gene cells with the The made by the in a of and in with human to with in cells in viability viability using a and in to a of in the cell of the cells in a with for to the of the using the using the to the by the with a of to from promoters. using the from cells the using the using from to the by the The for of the and in a to using a rate of the of and the for the an for of efficiency using the where cells in and the on the high for a of using the The proteins to a using the of the using the in in with using the using the The in this and and of for by cells using and using the for using using the with a to cell the and with cells for using a The cells using a and using in for while for The and the using and using using the with for using cells and in growth and on using the using cell and that we maintaining cell cells in cell The for the and in for on to the cells with for cell and cells with the a of cells for in the of to in the of cells and in to for cell the using the using and and from from in to nanoparticles to in in in and as in A. J.M. of lipid nanoparticles for in and in of PubMed Scopus Google Scholar a in with in an of a of using the of the system, both the and from a from their promoters. This a by the and are in a a rate of The of the and the the nanoparticles in to the of in The with up to and using a for The and to the nanoparticles for This for a of The nanoparticles a and the of the nanoparticles the The to a of the nanoparticles in the of to of the in in in nanoparticles containing by an PubMed Scopus Google Scholar and of the nanoparticles using and by on a in for both and and in five of and while in of order to the in and to using and using an and of in cells as in et C. 2020; PubMed Scopus Google Scholar cells using containing a for cells by to a of data are as and are of a of two using of using for using the while using the IntroductionOver recent years, many advances have been made in cancer therapy, including the development of targeted inhibitors and immunotherapies with greatly reduced adverse effects. Despite this, the current 5-year survival rate among all cancers is only 68%, dropping to under 10% for certain cancer types, including pancreatic cancer and glioblastoma.1Howlader N, Noone AM, Krapcho M, Miller D, Brest A, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, et al. (eds). SEER Cancer Statistics Review, 1975-2018, National Cancer Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2018/, based on November 2020 SEER data submission, posted to the SEER web site, April 2021.Google Scholar The resistance of these high-risk cancers to current therapies begets a need to develop novel treatment modalities to improve patient survival and quality of life. Several unique cancer treatment strategies have emerged by repurposing proteins found in viruses and bacteria to promote cell death, including the human herpes simplex virus thymidine kinase (HSV-tk) suicide gene and the CRISPR-Cas9 nuclease. Although both of these proteins have shown promise as treatment options for various cancers, there is a growing need to direct tumor-specific expression of these proteins in order to minimize the risk of off-target toxicity and, in the case of Cas9, off-target mutations.The hypoxic microenvironment offers a unique opportunity to target these exogenous proteins to regions of the tumor where the most aggressive and treatment-resistant cancer cells often reside. Solid tumors account for approximately 90% of all human malignancies.2Hematopoietic Cancers.in: Mak T.W. Saunders M.E. Jett B.D. Primer to the Immune Response. Academic Cell, 2014: 553-585Crossref Google Scholar As a result of vascular abnormalities that lead to low intratumoral blood flow, up to 50%–60% of locally advanced solid tumors develop areas of low O2 (<10 mmHg) partial pressure compared with their surrounding tissues.3Vaupel P. Mayer A. Hypoxia in cancer: significance and impact on clinical outcome.Cancer Metastasis Rev. 2007; 26: 225-239Crossref PubMed Scopus (1637) Google Scholar, 4Bernauer C. Stella Man Y.K. Chisholm J.C. Lepicard E.Y. Robinson S.P. Shipley J.M. Hypoxia and its therapeutic possibilities in paediatric cancers.Br. J. Cancer. 2020; 124: 539-551Crossref PubMed Scopus (16) Google Scholar, 5Höckel M. Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects.J. Natl. Cancer Inst. 2001; 93: 266-276Crossref PubMed Scopus (2131) Google Scholar This hypoxic state has been associated with increased tumor aggressiveness and resistance to current therapies.6Dhani A. M. The clinical significance of hypoxia in human PubMed Scopus Google Scholar all the proteins by hypoxic and their are the most the expression of is with a of approximately as a result of J. M. of factor is by an the Natl. A. PubMed Scopus Google factor in PubMed Scopus Google Scholar to within the of target including vascular endothelial growth factor containing copies of HRE sequences have been shown to specific gene expression in This gene expression has been in including hypoxia-specific cells and P. M. et cells and in solid PubMed Scopus Google M. of and in hypoxic tumor Google Scholar hypoxia-specific gene expression has been explored for the suicide gene gene using thymidine kinase and in and in PubMed Scopus Google J.M. of a hypoxia-responsive vector for tumor-specific gene PubMed Scopus Google Scholar we to improve on the and to this, regulation has been to the CRISPR-Cas9 to achieve specific expression of the CRISPR-Cas9 a unique opportunity to for tumor survival for no inhibitors By using HRE-driven we to cancer cell in hypoxic of the for is the of of and CRISPR-Cas9 in has been with levels of using A. and with the of suicide for targeted cancer Rev. PubMed Scopus Google Scholar, M. J. P. P. J. gene with by for with a PubMed Scopus Google Scholar, M. P. E.Y. and its PubMed Scopus Google Scholar significant for of CRISPR-Cas9 from the of the and the to direct in specific cell for in advances and PubMed Scopus Google Scholar the of therapies based on both and to virus 2020; PubMed Scopus Google Scholar are an to The promise and of in for PubMed Scopus Google Scholar with of in A. A. et of nanoparticles for 2020; PubMed Scopus Google Scholar, J. J. M. et for in the 2020; Scopus Google Scholar, J. C. J. et of lipid nanoparticles and in PubMed Scopus Google Scholar nanoparticles in a and to and therapeutic including and M.E. nanoparticles for Rev. 2020; PubMed Scopus Google Scholar, J. A. for cancer PubMed Scopus Google Scholar, nanoparticles for targeted from to J. PubMed Scopus Google Scholar, of CRISPR-Cas9 for specific 2020; PubMed Scopus Google Scholar, A. J.M. of lipid nanoparticles for in and in of PubMed Scopus Google Scholar many are made in order to the of to tumor A. et using targeted lipid nanoparticles for cancer 2020; PubMed Scopus Google J. M. of CRISPR-Cas9 and for tumor-specific gene with 2020; Scopus Google Scholar are to deliver cargo to the and a of for off-target nanoparticles gene from to clinical PubMed Scopus Google et in of nanoparticles for endothelial cell gene Natl. A. PubMed Scopus Google Scholar on these we to direct expression of and to and off-target the we on the development of both suicide gene and CRISPR-Cas9 therapeutic that expression in order to minimize off-target this we these from a promoter in with five copies of the HRE derived from the human using both hypoxia and we have cancer cell using two gene

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