Adding recombinant AAVs to the cancer therapeutics mix.

Introduction

Cancer is a collection of diseases caused by uncontrolled growth of damaged cells and is the second leading cause of death behind heart disease in the USA for women aged 40–79 years and men aged 60–79 years. 1 It is estimated that in 2022, 1.9 million new cases of cancer will be diagnosed, and more than 609,000 people will succumb to the disease. 1 Worldwide, cancer is a main cause of death, with estimates predicting case numbers around 29 million with 16 million deaths by 2040. 1 , 2 , 3 , 4 These numbers demonstrate the societal, personal, and economical impact cancer has on the world population. Overall, cancer diagnoses have increased, yet early detection and improved genetic analyses and targeted therapies have met this challenge with improved survival outcomes for several tumor types. Nevertheless, once cancers metastasize, the 5-year survival rate of patients dramatically drops, and cancers become more difficult to treat. Therefore, new, and more effective treatments are needed to continue to improve outcomes for patients with cancer. Moving forward, it is imperative for researchers to develop effective and affordable treatments that target and prevent cancer development early, and in the case of later-stage disease, we need therapeutics that are potent, specific, and able to prevent metastasis and recurrent cancer development. Gene therapy treatments designed to target mutated DNA sequences and correct them to restore proper function are being explored and are having a positive impact on multiple disease outcomes, including cancer. 5 , 6 , 7 , 8 , 9 , 10 , 11 In this review, we provide a brief overview of the cancer therapeutics landscape before focusing on the various types of gene therapy yielding encouraging results in preclinical and clinical settings for cancer, including advantages and limitations of these therapies. Finally, we concentrate on the biology and application of adeno-associated viruses (AAVs), one type of viral gene therapy with the potential to make a major impact on the cancer field due to their versatility, low immunogenicity, and long-lasting effects. 12

Current state of cancer therapeutics

Genomic sequencing of tumor biopsies has transformed cancer care and drug development. In the past, chemotherapies, radiation, and surgery were the best options for cancer patients, treatments that while effective to a certain degree, were potent and broad, causing side effects and potentially negatively affecting healthy cells as well as cancer cells ( Table 1 ). With genomic sequencing, physicians and researchers are able to identify driver mutations and druggable targets in specific patients’ tumors that allow for targeted therapies for cancers, improving responses and extending survival rates. 32 , 33 As described in Table 1 , the research into driver mutations and specific oncogenes has manifested clinically in a range of targeted therapies, such as small molecule inhibitors, antibodies, and immunotherapy that block these oncogenic pathways, as well as the various gene therapies being tested in myriad tumor types.

As a major trend in the cancer therapeutics field and a result of tumor sequencing, cancer immune therapy has made strong inroads in improving treatments and outcomes of patients. Currently, the main types of immune therapy that are making a great impact are the cellular immunotherapy, tumor-infiltrating lymphocytes (TIL), and immune checkpoint inhibitors (ICIs). 25 TIL therapy involves isolating lymphocytes that are present in a tumor mass and identifying in vitro which subsets of lymphocytes are best at targeting tumor cells via recognition of tumor-specific antigens. 34 , 35 Almost 100 clinical trials involving TILs and a wide assortment of solid tumors are currently in phase II or III, with documented success in treating melanoma and non-small cell lung cancer (NSCLC). 21 , 34 , 35 ICIs are a class of therapy that aims to activate anti-tumor T cells to prevent the cancer from avoiding immune destruction. 7 The main targets of ICIs are the cell surface proteins cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death-1 (PD-1), and PD-1’s ligand, PDL-1. 25 , 26 , 27 , 36 Inhibition of these three proteins blocks the inhibitory signal or death signal being induced by the cancer cells, which then facilitates T cell-mediated destruction of the cancer. Ipilimumab, pembrolizumab, and avelumab are some examples of FDA-approved ICIs having a positive impact in various solid tumors. 26 , 27 , 36

This process of genomic sequencing has also advanced subtyping of tumors, another detail that guides physicians and scientists in selecting treatment regimens and designing studies to expand cancer therapeutics research. For example, a monumental impact has been made in treating lung cancers with genomic sequencing. Meador and colleagues spent years identifying a subtype of NSCLC driven by EGFR (epidermal growth factor receptor). 18 , 19 Even within this subtype, different mutations in the EGFR gene occurred in certain tumors in response to first-generation EGFR tyrosine kinase inhibitors (RTKi), illuminating mechanisms of targeted drug resistance and methods of treating patients most effectively with this NSCLC subtype. While several new RTKi’s and monoclonal antibody therapeutics are being explored and having some success in treating drug-resistant NSCLC, many of these tumors develop new mutations that confer resistance. The majority of genetic aberrations that are driving these documented resistances occur in exon 19 as a deletion, an L858R point mutation in exon 21, and various point mutations as well as an in-frame insertion in exon 20 of the EGFR gene, suggesting targeting the EGFR DNA directly with gene therapy or in combination with standard therapeutics might be the best way to treat these NSCLCs. 18 , 19 , 34

One of the most well-known and frustrating oncogenes in the cancer field is KRAS. Deemed an undruggable target decades ago, this gene has been the subject of multitudes of research, and targeted therapies directed at this protein are being studied for use in several tumor types, such as lung cancers, pancreatic cancer, and colorectal cancer. 37 Another tumor type that has benefited greatly from genomic sequencing to employ and guide targeted therapies is T cell acute lymphoblastic leukemia (T-ALL). With the identification of multiple subtypes of T-ALL by genomic analyses, each was shown to rely on distinct driver mutations and activated signaling pathways. 13 , 38 These genomic subclassifications shaped how patients needed to be treated in the clinic. Indeed, drugs targeting specific pathways such as NOTCH, Jak/Stat, and PI3K are being researched and tested in T-ALL patients. 14 In these few cancer examples, a common thread is that the oncogenic pathways driving these diseases are highly mutagenic, have been difficult to target effectively with current drug types, and they influence several other pro-oncogenic pathways. New and specific therapies that correct the mutations at the DNA level such as viral-based gene therapies could be incredibly impactful as a treatment. Indeed, reported therapeutic regimens being tested pre-clinically that contain a gene therapy like AAV and established drugs like immunotherapy or chemotherapy are showing promise in cancers such as ovarian, hepatocellular carcinoma, and triple-negative breast cancer. 5 , 7 , 9 , 26

Additionally, drug repurposing for cancer treatment has been explored for several reasons: cost effectiveness, FDA approval already given for the drug, and immediate availability for patients. 39 One well-known example is thalidomide, an immunomodulatory drug whose original purpose was to treat skin pathologies like leprosy. Now, this drug is used to treat multiple myeloma usually in combination with the proteasome inhibitor bortezomib and/or the steroid dexamethasone. 30 Some success has been achieved with other classes of drugs, such as the anti-obesity drug orlistat, and some of the avermectin-type drugs. 40 , 41 Clinicaltrials.gov reports drugs such as chlorpromazine, metformin, and mebendazole being tested in patients for glioblastoma, prostate cancer, and hepatocellular carcinoma (HCC), respectively. While these drugs being explored for repurposing in cancer would be beneficial, their multi-targeting characteristics carry the risk of off-target effects and side effects that could be problematic for patients. Even with these improvements in the field, a percentage of patients still experience recurrent disease or have cancers that mutate and no longer respond to treatment regimens that were previously successful. Moreover, once cancers metastasize, spread to distant sites in the body, some treatment options are no longer effective at preventing growth. 1 Therefore, new types of drugs and treatment regimens are needed to continue to reduce the cancer death rate, prevent metastatic disease, and provide hope for those diagnosed with any type of cancer. Hence, gene therapy is being explored as a viable, long-term corrective option for treating several cancer types. It is true that non-viral-based therapies, such as cell therapy and nucleic acid-based therapies, have shown success in certain tumor types, especially hematological cancers. 15 , 25 , 42 , 43 , 44

Non-AAV gene therapies for cancer

As previously stated, gene therapy is defined as treatment of a disease using techniques that alter the genes of cells with the hope of preventing or stopping disease progression. 45 This type of therapy aims to replace defective genes or mutated cells that are the cause of a disease or add in a new gene that can help fight the disease and improve the patient’s quality of life. 46 There are several types of gene therapy that have been used in the lab to study myriad of pathologies, and more of these gene therapies are being tested in clinical trials and approved for treating patients. Non-viral therapies, such as cell therapies and interfering RNA, as well as viral-based therapies are showing promising results in targeting not only rare disease but cancers as well.

Cell therapy

Cell therapy is a non-viral type of gene therapy that utilizes healthy cells or cells engineered ex vivo to perform a certain function and/or express certain proteins to combat cancer. 25 The most well-known version of cell therapy for cancer now is a T cell transfer therapy in the form of chimeric antigen receptor T cell (CAR-T) therapy. CAR-T cells are T cells that are removed from a patient’s body and manipulated in culture to better bind tumor cells and attack the cancer in the hopes of killing it. Several CAR-T therapies have been FDA approved to treat hematological cancers. One example, Kymriah (tisagenlecleucel), is used to treat B cell ALL and diffuse large B cell lymphoma (DLBCL). This CAR-T therapy recognizes CD19, a surface protein expressed at high levels on cancerous B cells and facilitates T cell expansion upon binding in order to kill the tumor. 42 , 47 , 48 In March of 2021, the CAR-T Abecma (idecabtagene vicleucel) was approved to treat refractory or relapsed multiple myeloma (MM), as it targets the elevated expression of B cell maturation antigen (BCMA) present on MM tumor cells. 49 In addition to hematological cancers, CAR-T therapy is being explored for uses in solid tumors like breast cancer and brain cancers. Two main drawbacks to CAR-T therapy relate to the specificity of the target and the potential for the side effect known as cytokine release syndrome (CRS). In the examples of Kymriah and Abecma, their targets, CD19 and BCMA, are not tumor-specific proteins. These surface proteins are also expressed, although at lower levels, on healthy B cells and plasma cells, implying that healthy cells could also be targeted and killed by the therapy. CRS is a potentially severe inflammatory response that is reported to occur in some patients that receive CD19 CAR-T therapy. 50 Fever, abnormal heartbeat, and hypotension are common signs, with most severe cases leading to capillary leak syndrome, low blood pressure, organ failure, and death. 51 A range of interleukins and interferon-gamma are just some of the cytokines that are activated in CRS. 51 , 52

Nucleic acid-based cancer therapies

Along with cellular therapies, researchers and physicians are testing other non-viral gene therapies for their efficacy against several cancer types. A large class of these therapies are based on nucleic acid structures and are designed to target RNA to prevent translation or to introduce new DNA into tumor cells to prevent growth or induce a host immune response. A key component of these nucleic acid-based gene therapies is the vehicle in which the therapy is encapsulated to facilitate stability and successful delivery to the target. Nanoparticles, liposomes, and microbubbles are just a few of the carriers being researched for gene therapy delivery. 15 , 43 , 44 Groups report experimental success of DNA delivery of melanoma-specific antigens and IL-12 to treat melanoma lung metastases and colorectal cancer, respectively. 53 , 54 Similar methods have been tested with the cargos being interfering RNA molecules (RNAi): small interfering RNA (siRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), and antisense oligonucleotides (ASO). These RNAi molecules each enact their inhibition in slightly different ways through base pairing at distinct stages of RNA processing, with the goal of silencing gene expression. 55 , 56 siRNAs are double-stranded RNA molecules designed to be 100% complementary to the gene target. Therefore, siRNA is highly specific and potent. miRNAs are single-stranded natural RNA molecules that are produced by cells in the body. miRNAs are somewhat promiscuous as certain miRNAs can regulate expression of several genes. In cancer research, many groups have identified miRNAs that are tumor suppressive and those that promote cancer development, which has guided researchers in using miRNAs as therapy. 57 , 58 shRNAs are double-stranded RNA molecules that are designed to undergo the biological processing to siRNA using cell machinery. shRNAs also allow for a greater chance of stable expression and suppression of target gene activity. ASOs are single-stranded RNA or DNA constructs designed to bind mRNA via complementary base pairing to regulate genes at the transcription level through physical steric blockade of producing mature mRNA or inducing RNase-H-mediated processing of the mRNA. 59 , 60 , 61 Typically, ASOs are 15–30 bps and contain chemically modified backbones to improve stability, protection from nucleases, and enhance efficacy. 59 In the context of cancer biology, ASOs have been tested with moderate success in oral squamous cell carcinoma, Kaposi’s sarcoma, and targeting a variety of angiogenic oncogenes; yet, no ASO is FDA approved to treat cancer to date. 60 Difficulties and concerns with RNAi therapies are mostly related to dosing, efficacy of vector, low delivery of RNA molecule, renal clearance, and off-target effects due to the selected carrier. 7 , 59 , 60 , 61

Oncolytic viruses

Another group of gene therapy vectors being researched for their efficacy against cancer are a group of viruses deemed oncolytic viruses. This class of viral-based gene therapy encompasses a range of different virus families. 62 The common thread among them is that they preferentially infect tumor cells, replicate within the cells, arrest their growth, and induce some form of tumor destruction. Depending on the design of the engineered virus or the biological properties of the unmodified virus, tumor destruction manifests most often as apoptosis, a focused immune response, or a reduction in tumor vasculature. Adenovirus, herpesvirus, measles virus, and vaccinia virus are among the types being tested in preclinical and clinical research with documented success in solid tumors such as HCC, melanoma, and glioblastoma. Excitingly, ovarian cancer and neuroendocrine tumors (NENs) are newer focuses for the field. The oncolytic adenovirus dl 922-947 is reported to synergize with the beta-blocker carvedilol and increases survival in a mouse patient-derived xenograft (PDX) model of ovarian cancer. 63 Inoue and colleagues report treatment with the vaccinia virus mpJX-594 enhances anti-PD1 immunotherapy in pancreatic NENs. 64 Additionally, rodent protoparvoviruses (PVs) are also being explored for their efficacy against many types of cancers. Specifically, H-1 parvovirus (H-1PV) and minute virus of mice have shown promise both translationally and at early clinical stages of research. These PVs contain a ssDNA genome and are reported to preferentially infect tumor cells. 65 They kill tumor cells mainly through activating DNA damage responses, causing reactive oxygen species, and inducing apoptosis. 66 , 67 , 68 These mechanisms are observed in cancers that are very aggressive and difficult to treat: glioblastoma, gliomas, and pancreatic ductal adenocarcinoma. 69 , 70 , 71 , 72

As a group, oncolytic viruses are quite promising, as evidenced by the FDA approval of Imlygic, a weakened herpes simplex virus (HSV-1) used to treat melanoma present on skin or in lymph nodes ( Table 2 ). While the exact mechanism of action is not fully understood, this HSV-1 is injected directly into the tumor mass and induces a localized immune response to the tumor via GM-CSF production and tumor lysis. 79 Yet, oncolytic viruses as a therapy also present with certain disadvantages. As they are a diverse group of vectors, production details and purity will depend on which carrier is used. Due to this, high titer and details in protocol issues can arise in moving to clinical studies. 80 , 81 Given the mechanism of action of these viruses, off-target effects or amplified immune responses to the treatment are a concern. Flu-like symptoms and injection site issues such as pain have been reported. 81 , 82 , 83 This reinforces the importance of design and tumor specificity. Also, activation of the immune response to some of these viruses reduces the amount of oncolytic virus (OV) that can target the tumor, thereby compromising the efficacy of the OV therapy. 83 Some reported mechanisms include macrophages engaging and clearing viral carriers, innate immune activation via Toll-like receptors caused by viral RNA or CpG islands, as well as the humoral immune activation through production of neutralizing antibodies toward the viral capsid. 62 , 65 , 81 , 84 , 85 Currently, most oncolytic viruses are injected intratumorally, which excludes hematological cancers or cancers in early stage of disease that have not formed a mass. 62 , 81 , 82 , 83 Of note, Reolysin, a reovirus-based oncolytic viral gene therapy, is being tested for efficacy against MM among many solid tumors ( NCT02514382 ).

Recombinant AAV gene therapy

AAV therapy, a viral gene therapy, has great possibility for improving therapeutics in the cancer biology field and effectively targeting several cancer types due to its low immunogenicity, minimal side effects, long-term expression of the transgene, adaptability, and no direct link to causing human diseases. AAVs belong to the parvoviridae family and are a single-stranded DNA virus with a genome of approximately 4.7kbs. 86 , 87 The genome of the wild-type AAV encodes Rep and Cap genes, Rep encoding for viral replication, packaging and genomic integration, and Cap encoding for the structural proteins VP1, VP2, and VP3 that constitute the viral capsid ( Figure 1 A). 93 The AAV genome is buttressed by two inverted terminal repeat sequences (ITR), facilitating packing of the AAV genome into the AAV molecule. 94 If a helper virus is present, the AAV is able to replicate. With no helper virus, AAVs become latent and remain in episomal form. While rare, AAV genomic integration into host chromosomes has also been reported. The genome of AAVs can be manipulated and changed to create recombinant AAVs (rAAVs); this is the basis of AAV-based gene therapy. These rAAVs contain the two ITRs along with a specific promoter sequence, a gene of interest driven by that promoter, and a poly-A tail sequence that supports nuclear export of RNA and translation ( Figure 1 B). rAAVs are attractive for therapy for several reasons. Along with multiple tested and reliable promoters that can drive expression of the transgene of interest, rAAVs are minimally immunogenic and can infect dividing and non-dividing cells with the resulting genetic changes being long-term, making them widely applicable to many disease models. Also, AAV infection is not directly linked to causing any sort of pathogenesis, demonstrating a safety profile that suggests the virus would cause limited side effects. These details are emphasized by the increase in the number of clinical trials in the USA that include an AAV over the past 16 years. As highlighted by D. Kuzmin and co-authors, the various AAVs are now being used to target many different diseases through specific tissue tropism ( Figure 1 C). 95 On the other hand, the relatively small genome (4.7 kb), a rare possibility of rAAV integration into the host genome, and the potential for people to have natural neutralizing antibodies against certain AAVs serotypes are some disadvantages of this gene therapy vector. 88 , 89 , 96 , 97 , 98

As previously stated, AAVs exhibit relatively low immunogenicity compared with other vectors and are not directly linked to causing any human pathology. 7 , 9 , 99 , 100 These data suggest that several AAV serotypes are not inducing strong immune reactions in the host that could be detrimental to either the host’s health or the efficacy of the rAAV-based therapy. As many rAAVs are being designed and tested in several cancers to cause a controlled, focused response to target the tumor, it is key that the AAV itself does not influence the host immune system in a way that could compromise the effects of the therapy. 5 , 8 , 9 , 101 , 102 , 103 Even in reports of hemophilia A where a high dose of an rAAV driving expression of factor VIII is inducing immune responses, such as neutralizing antibodies toward the AAV capsid, strategies to dampen the immune response to the virus or enhance the efficacy of the rAAV are showing promise and could be broadly applicable. 97 , 104 , 105 , 106 Other types of viral vectors and nucleic acid-based methods of gene therapy being explored do not consistently exhibit these characteristics of low immunogenicity or no links to human pathology, making AAVs an attractive vehicle for gene therapy. Moreover, rAAVs convey long-term expression of the transgene and are versatile as their genomes are easily manipulated and altered to meet the needs of specific studies and pathologies. One of the first AAV therapies approved for use in patients was Luxturna, an AAV2 used to treat Leber’s congenital amaurosis by inducing expression of the RPE65 gene. 73 , 107 Spinal muscular atrophy, another rare disease, is treated with an AAV9 gene therapy that facilitates alternative splicing of the SMN gene to produce the lacking SMN1 protein in patients with this disease. 108 Both AAV treatments are widely successful and have catalyzed the AAV therapeutics field now being explored in diseases such as hemophilia, Duchenne’s muscular dystrophy, and cancer. 105 , 109 , 110 , 111 As cancer encompasses a heterogeneous group of diseases as well as single tumors containing intratumoral heterogeneity, the ability to tailor an AAV for a cancer type, specific tumor, or distinct cancer cell within a tumor is highly beneficial. Moreover, the long-term expression of the specific transgene conveyed by rAAV would provide a cancer therapeutic for patients with a longer half-life and could theoretically reduce frequency of hospital visits compared with regimens that contain other available treatments like chemotherapies, which require several rounds of administration due to their short half-lives, periods of no therapeutic intervention, and clinic appointments ( Table 1 ). 1 , 4 , 15 , 31 Translational studies from multiple researchers show that several cancer subtypes, along with cancer-associated stroma and the supportive cancer microenvironment, are currently being targeted effectively with rAAVs. 6 , 8 , 9 , 11 , 55 , 56 , 112 , 113 , 114 , 115

rAAVs targeting primary tumors

rAAVs are a revolutionary therapeutic avenue being explored for specifically targeting primary tumors. These viral vectors are being designed to correct oncogene expression or restore tumor suppressor activity, to induce cell death, block angiogenesis, or to activate anti-tumor host immunity ( Figure 2 ). Multiple AAV subtypes, including AAV6 and AAV2, have demonstrated success in infecting and reducing proliferation and growth of multiple cancer cell lines as well as the NeuT mouse mammary tumor model. 55 , 116 M. Trepel et al. reported that an rAAV9 vector encoding HSV-TK, a suicide gene that creates a cytotoxic metabolite of the drug ganciclovir (GCV), along with a sequence that reduced cardiotoxicity, reduced mammary tumor growth and eliminated negative effects on ejection fraction and cardiac damage of the polyoma middle tumor antigen mouse model. 117 There is also documented success of rAAVs successfully targeting HCC. Dhungel and co-authors report multiple AAV-based therapies being explored to target HCC. 6 Many of the early studies using rAAVs to target HCC drove expression of genes that caused apoptosis in these tumors. HSV-TK and TRAIL, a pro-apoptosis factor, are two of the most popular therapeutic genes studied in HCC. 115 , 118 , 119 Upon treatment with GCV, tumor cells that express HSV-TK will undergo apoptosis once the metabolite GCV-3P is made. Neighboring tumor cells are affected, which leads to tumor death. Regarding AAV-driven TRAIL expression, the hTERT promoter has been shown to be specific in targeting tumor cells and causing apoptosis both in vitro and in vivo mouse models of HCC. In recent studies, scientists have expanded rAAV target genes to target HCC via other mechanisms. L. Yin and colleagues demonstrated that an AAV3 vector driving expression of either miR-26a or miR-122 was effective at reducing growth of Huh7-FLUC cells in NSG mice. 58 These miRNAs are known to target angiogenic pathways, Wnt/β-catenin, and certain cyclins. Given the tropism of several AAVs to liver, HCC is a viable, strong cancer target for AAV gene therapy. Indeed, AAV2 was shown in two mouse models of HCC development as well as human precision-cut liver slice experiments to preferentially infect tumor tissue compared with nonmalignant liver area. 120 Additionally, using an AAV3 vector to drive expression of pyruvate dehydrogenase E1α led to cell death in human HCC. 121

Several other cancer types, including rare and aggressive tumors, are also being studied with rAAVs. H. Li and co-authors showed that an AAV2/9 vector controlling expression of an shRNA that silenced the oncogene FOXP1 greatly reduced osteosarcoma growth in a PDX model over a 5-week time course ( Figure 3 A). 127 As this tumor type is rare and treatment options are limited, rAAV therapy could be a great new resource for effective therapies. Moreover, modifications of capsid proteins and the addition of specific conjugates to certain AAVs, like AAV6, changed its transduction capabilities in infecting myriad of cancer cells lines including PC3 prostate cancer cells and T47D ductal carcinoma cells. 116 Prostate (PCa) and breast cancer (BCa) are the most diagnosed cancers in men and women, respectively. 1 In an innovative study, Cripe and colleagues utilized rAAV8 and AAVrh74 to create enhanced, AAV-based versions of blinatumomab, a bispecific CAR-T antibody approved to treat CD19+ ALL. 28 , 128 Their rAAVs extended the half-life of the antibody as well as converted the drug to be inducible in one iteration of the therapy; each modification resulted in improved survival of NSG immunocompromised mice injected with Raji lymphoma cells. 28 This report adds to the plethora of applications for rAAVs in cancer biology. Regarding rarer cancers, S. Lee and H-J. Ahn demonstrated that modifying AAV2 to interact with EpCAM, a surface protein highly expressed in ovarian cancer, was effective at delivering an shRNA targeting EGFR signaling, significantly reducing ovarian tumor growth in Balb/C-Nude mice. 129 AAV2 without this EpCAM modification was ineffective at blocking tumor growth, demonstrating the wide range of ways to make AAVs effective in targeting tumors at the primary site. Similar to early research with HCC, many of the gene therapy studies regarding these tumor types have centered on driving expression of suicide genes by promoters like GRP78 and PSA for PCa, and ErbB2 and MUC1 for BCa. 10 , 11 , 56 , 61 , 113 An AAV9 construct using either the non-specific chicken beta actin promoter or the neuron-specific enolase promoter driving expression of soluble TRAIL demonstrated a retardation of a PDX orthotopic glioblastoma tumor model. 122 The breadth of cancers that are being successfully targeting in translational studies, especially those used in PDX models, suggests an exciting and hopeful future in cancer research.

rAAVs targeting metastatic lesions

The documented translational research successes of rAAVs in cancer studies infer that this viral gene therapy could be groundbreaking in treating a catastrophic event that occurs during many patients’ battles with the disease, metastasis. Regardless of tumor type, once metastasis occurs and the primary tumor spreads to distant secondary sites, the 5-year survival rate of the patient dramatically decreases, emphasizing the need for early effective treatments and metastasis-specific therapies that prolong survival. 1 , 4 , 10 , 22 , 130 , 131 , 132 , 133 , 134 , 135 The cells of a tumor that metastasize to a secondary organ express a genetic profile that is different from the primary tumor, a characteristic of tumor heterogeneity that in some ways makes metastatic cancer its own distinct disease. 130 , 132 , 134 , 136 For example, several groups report specific secretome and genetic profiles of metastatic clones of cancer cell lines compared with the parental line, a biological trait that can be exploited by the engineering versatility of AAVs to specifically and effectively target subgroups of cancers located in either a primary or secondary site. 54 , 132 , 137 In breast cancer and lung cancer, COL6A1 is reportedly expressed at higher levels in metastatic tissue compared with non-metastatic tissue. 138 Work published by M.A. Blanco and colleagues identified plasminogen and other tissue repair genes compiling a bone metastatic profile of the triple-negative breast cancer (TNBC) MDA-MB-231 cells and the murine mammary tumor line 4T1s. Forced expression of these plasminogen genes in the non-bony metastatic SCP28 breast cancer cell line caused these cells to invade bone and produce osteolytic lesions. 139 Prostate cancer cells that express high levels of ESL-1, a protein involved in diapedesis and endothelial adhesion, exhibited enhanced bone metastasis. 137

Encouragingly, rAAVs being synthesized to target metastatic lesions are showing promise in many metastatic tumor models. For example, Y. Xia and associates demonstrated that treating mice with the Anc80L65 AAV encoding DNaseI under control of the human alpha-1 antitrypsin promoter significantly reduced colonization of circulating MC38 CRC cells in the liver of C57Bl/6 mice through induction of a host immune response. 123 Increased expression of this enzyme induced a CD45+ immune response in the metastatic liver lesions and reduced the amount of tumor-supportive neutrophil extracellular traps (NETs) in the liver tumor microenvironment. Consequently, this rAAV treatment reduced colonization of circulating MC38 CRC cells in the liver. Focusing on a different hallmark of cancer, pulmonary metastatic lesions of the aggressive 4T1 mouse mammary carcinoma model were targeted with an AAV2 vector that drove expression of VEGF-Trap, an antiangiogenic chimeric protein that prevents angiogenesis by binding VEGF-A as a decoy receptor. Not only were the number and size of lung metastatic lesions reduced, but the primary tumor size was also decreased because of this VEGF-Trap gene therapy ( Figure 3 B). 124 Importantly, VEGF-Trap expression maintained throughout the in vivo studies after a single dose. A third example of successful application of rAAVs in targeting metastatic disease is reported in bone. 130 Bone metastases remain a devastating clinical outcome of several commonly diagnosed cancers including breast and prostate. Work from Y. Yao and colleagues reveals that AAVs could be an effective treatment in the future for these types of lesions. 140 They used an rAAV2 driving expression of Kringle 1 domain of human hepatocyte growth factor, a known antiangiogenic and antiproliferative protein, to prevent osteolytic lesion development in vivo using a bone-tropic MDA-MB-231 human TNBC cell line. As metastatic disease results in extremely worse prognoses for cancer patients regardless of tumor type, building upon results such as these will be critical in future AAV gene therapy work.

rAAVs targeting the tumor microenvironment

It is well-documented that non-cancerous cells, extracellular matrices, and physical properties of various organs and regions of the body facilitate tumor development and metastasis. 21 , 130 , 131 , 133 , 135 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 Because of these data, several conventional cancer therapeutics indeed have been designed to block signaling of and interactions between stromal cells such as cancer-associated fibroblasts, vasculature, and pro-tumor immune cells, and tumor cells with varying successes. 22 , 152 , 153 , 154 , 155 , 156 , 157 In the context of gene therapy, rAAVs are also being designed to target these types of tumor-supporting cells. As previously mentioned, the rAAV utilized by Y. Xia and co-authors to drive expression of DNase I in order to target liver metastatic lesions of the MC38 CRC cell line was successful via targeting NETs and increasing the population of anti-tumor CD8 T cells in the liver lesions ( Figure 4 ). 123 rAAVs containing sPD-1 have been documented to alter the host immune response in mouse models of cancer. Elhag et al. showed increased anti-tumor cytotoxic T lymphocyte infiltration in a hepatoma mouse model, leading to increased survival. 158 Similarly, Z. Tan and colleagues published in a mesothelioma mouse model that an rAAV-DJ serotype driving sPD-1 fused with a common oncogene, TWIST1, and increased CD8+ T cells in the tumor mass, leading to increase survival. 159 rAAV2 has also been reported to infect cancer-associated vasculature in the NeuT mouse mammary tumor model, reducing vascular length and increasing apoptosis in tumor-associated “endothelial cells (ECs).” 55 Along with altering the immune response to tumors, A. Volak et al. showed that an rAAV from their group, deemed exo-AAV, was able to cross the blood-brain barrier and be modified to infect various stromal cells in an aggressive glioblastoma mouse model, including reactive astrocytes and tumor-associated microglia. When the exo-AAV was designed to target astrocytes using glial fibrillary acidic protein promoter, a modest survival increase was observed along with an increase in tumor mass death. 103 Indeed, these preclinical benefits of targeting the tumor microenvironment demonstrate even more possible applications for rAAV gene therapy to impact various cancers, especially those in need of new treatments, in the clinic.

Intriguingly, AAVs infect cells by initial contact with a range of cell-surface receptors and integrins. For example, AAV2 is known to interact with heparan sulfate proteoglycan, laminin receptor, MET, and integrins containing the a v subunit. 46 , 96 , 160 , 161 , 162 Overexpression of many of these proteins and their ligands is a driver of cancer growth and metastasis, as well as being expressed by myriad supporting cells that fuel cancer growth. These data infer that cancer cells and their supportive stroma are prime targets for rAAVs infection. The versatility and malleability of rAAVs via transgene expression, capsid serotype, and the promoter sequence that drives the transgene can be synthesized to effectively target cancer, specifically those that have not had much progress in therapeutics in recent history.

rAAV design and specificity in targeting cancers

The effectiveness of rAAVs in targeting cancer depends on multiple details: the specific AAV capsid protein, the encoded promoter, and the transgene being driven by the specific promoter. 163 These components of the AAV can be manipulated to create an adaptable range of effective AAVs to target many aspects of cancer development, growth, and metastasis in various tissues.

There are 13 natural serotypes of AAV, and each has distinct biological properties, including tissue tropism. 89 , 90 , 164 For example, AAV7, AAV8, and AAV9 have a reported tropism for liver and are being used to target diseases like hemophilia. 105 , 109 Other AAVs, such as AAV1, infect heart and skeletal muscle regularly, and AAV2 and AAV3 infect the retina as well as the liver. 86 , 165 The receptors that certain AAVs interact with to enter cells varies, and this is related to the specificity seen in certain cells and tissue types. Scientists have also been able to engineer AAVs with specific capsids that contain a mixture of several natural AAVs, hybrid AAVs, modifications on surface proteins that make the capsids bind receptors with greater affinity, or AAVs that combine traits from different animal sources such as rats, mice, and goats. 12 , 89 , 166 , 167 Interestingly, the AAV tropism of non-transformed tissues does not always match the tropism profile of AAV infection of tumor tissues. AAV3 is reported to infect liver cancer cells at a greater rate than healthy hepatocytes. 58 , 121 This disparity is actually advantageous, as a key point of cancer therapy should be to spare as many healthy cells as possible and target only tumor cells. Moreover, our group is exploring chemical modifications of AAV capsid proteins, which is illuminating different profiles of AAV organ tropism and infection patterns. 91

A review by Montaño-Samaniego and colleagues superbly explains how AAV design should be engineered with the promoter in mind to maximize efficiency. 61 Genes of the specific tissue affected by the tumor, genes that are specific to cancers across types, and/or genes specific to a particular tumor type or subtype can be used as the promoter encoded by the AAV to direct the virus. There are several promoters considered ubiquitous; this refers to the non-specific nature of the gene from which the promoter is derived and the potential for broad, high levels of transgene transcription and translation. Some examples include CMV, CB, EF1α, and TBG. While reliable to induce expression of the transgene, the drawback in using these types of promoters is the potential in altering stromal, non-cancerous cells. This group of promoters contrasts with tissue-specific promoters used to induce more controlled, targeted expression of the transgene of interest. For example, rAAVs designed to target hemophilia, a set of hematological clotting disorders, contain the liver-specific transthyretin promoter. 96 , 161 , 162 , 163 This distinct promoter relegates transgene expression of the absent clotting factor protein ( FVIII for HemA or FIX for HemB) to hepatocytes and the organ where these clotting proteins are normally synthesized. 98 , 161 , 168 , 169 , 170 This concept can be expanded to using cancer-specific promoters from genes that are generally overexpressed in several tumor types, as well as tumor-specific promoters, which would be a gene unique to the particular tumor target of the rAAV. Some effective promoters currently being explored include hTERT, AFP, and ERBB2. 113 , 125 , 171 One of the goals of rAAV therapy is to be as targeted and specific to the cancer mass as possible, and the selection of the promoter is a key feature in achieving tumor specificity.

Equally important in engineering rAAVs for cancer is the transgene being driven by the promoter in the vector. As stated earlier, myriad genes are being explored for efficacy against several cancers. These transgenes demonstrate varying levels of effectiveness against primary tumor lesions as well as some metastatic growths that occur during progression. Pro-apoptotic genes such as TRAIL and miR26a, cytokines that stimulate anti-tumor immunity like IL-27 and IL-15, and antiangiogenic proteins are some examples of in vitro and translational research successes. 101 , 102 , 124 , 172 , 173 , 174 The AAV serotype and the promoter will control the distribution and effects of the transgene in the target tumor and tissue, giving rise to a potential revolutionary set of tools to treat cancer and improve patients’ lives. rAAVs that are specific for tumors or common metastatic sites could be designed as prophylactics for patients recovering from a primary tumor or those at high risk of developing cancer. Moreover, rAAVs can be used as a long-term treatment for established cancer lesions and preventing recurrence, as an attractive, beneficial feature of this developing therapy is the long-term effects observed after one dose observed in diseases such as Leber’s congenital amaurosis, hemophilia, and spinal muscular atrophy. 73 , 105 , 108 , 109

Combination therapies with rAAVs for cancers

In addition to targeting the many cells and factors that comprise and influence cancer progression, approaches combining rAAVs with current cancer therapies are being explored pre-clinically. 5 , 7 Many of the combinations include a chemotherapeutic agent such as platinum-based drugs, 5-fluorouracil, and doxorubicin that supplements the rAAV gene therapy that is targeting a specific aspect of the tumor. 5 , 16 These combinations apply cell cycle stress on the tumor cells that lead to toxicity and cell death. Inhibition of angiogenesis via encoding expression of bevacizumab or inducing cell death pathways through expression of pro-apoptotic gene TRAIL are some rAAV designs that have shown enhanced cytotoxic stress in tumors, leading to more death when combined with a standard chemotherapy. 17 , 23 , 175 While more details on the specific mechanisms of action need to be determined for these combination treatments, the results are promising and add to the potential impact rAAVs could have in the cancer field.

Conclusion

Cancer is a devastating group of diseases that continues to have a massive negative global impact. Despite improved survival rates through early detection, better care, and improved therapies, millions will succumb to the disease. Gene therapy is having a positive impact on several diseases in the clinic. rAAVs, a virus-based gene delivery platform, could be a groundbreaking new group of effective therapies for targeting cancers of any type. rAAV’s versatility, ability to be tailored to specific tissues and cells, and low immunogenicity are traits that epitomize the ideal cancer treatment: specific, potent, and with potentially limited side effects. With cancer patients living longer, and dormant secondary lesions having the ability to manifest months to years after an initial treatment, the long-term effects of a successful, targeted rAAV therapy could change the way cancers are treated, making them more of a manageable disease than an outright, eventual death sentence. The limitations and hurdles of using rAAVs, namely natural immunity to certain serotypes present in individual patients, a relatively small genome size (4.7–5 kba), documented production of neutralizing antibodies to various serotypes, low chance of genomic integration into the host genome, and possible off-target infection of certain tissues or cell types based on capsid serotype, promoter, or transgene, will need to be a top interest of scientists and clinicians as research testing of rAAVs and cancer expands. As there are many aspects of cancer that need to be addressed clinically, current results of rAAVs and cancer are encouraging in the hope that rAAVs will be used to target cancer, cancer metastatic disease, and the myriad co-morbidities that arise specifically from the disease. Although more translational and preclinical research is warranted to ensure safety of the many different rAAVs that can be designed for cancers in the clinic, the possibilities of this viral gene therapy are limitless and could direct the future of cancer therapeutics, especially for metastatic cancers, residual and relapsed disease, and rare cancers.

Acknowledgments

We thank the many members of the Xiao and Herzog laboratories and Dr. Ushashi Dadwal for several discussions surrounding AAVs and cancer. Funding sources include National Institutes of Health (NIH) R01HL130871 (W.X.), U54HL142019 (W.X.), and P01HL160472 (R.W.H., W.X.).

Author contributions

P.L.M. and W.X. conceived the concept for the review. P.L.M. wrote the manuscript and designed figures using BioRender.com licensed through IUSM Herman B Wells Center for Pediatric Research. R.W.H. and W.X. edited the manuscript and provided scientific guidance for the final draft. P.L.M. conducted revisions for the review. All members approved the final draft.

Declaration of interests

R.W.H. is editor-in-chief of Molecular Therapy and has served on scientific advisory boards for Regeneron Pharmaceuticals and BioMarin Pharmaceuticals. W.X. holds equity in the following: Ivygen Corportion and Nikegen LLC.

References

1. Siegel R.L., Miller K.D., Fuchs H.E., Jemal A. Cancer statistics, 2022. CA. Cancer J. Clin. 2022;72:7–33. doi: 10.3322/caac.21708.

2. Bray F., Ferlay J., Soerjomataram I., Siegel R.L., Torre L.A., Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA. Cancer J. Clin. 2018;68:394–424. doi: 10.3322/caac.21492.

3. Ferlay J., Colombet M., Soerjomataram I., Mathers C., Parkin D.M., Piñeros M., Znaor A., Bray F. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int. J. Cancer. 2019;144:1941–1953. doi: 10.1002/ijc.31937.

4. Siegel R.L., Miller K.D., Fuchs H.E., Jemal A. Cancer statistics, 2021. CA. Cancer J. Clin. 2021;71:7–33. doi: 10.3322/caac.21654.

5. Bower J.J., Song L., Bastola P., Hirsch M.L. Harnessing the natural biology of adeno-associated virus to enhance the efficacy of cancer gene therapy. Viruses. 2021;23:1205. doi: 10.3390/v13071205.

6. Dhungel B., Jayachandran A., Layton C.J., Steel J.C. Seek and destroy: targeted adeno-associated viruses for gene delivery to hepatocellular carcinoma. Drug Deliv. 2017;24:289–299. doi: 10.1080/10717544.2016.1247926.

7. Hacker U.T., Bentler M., Kaniowska D., Morgan M., Büning H. Towards clinical implementation of adeno-associated virus (AAV) vectors for cancer gene therapy: current status and future perspectives. Cancers (Basel) 2020;12:E1889. doi: 10.3390/cancers12071889.

8. Luo J., Luo Y., Sun J., Zhou Y., Zhang Y., Yang X. Adeno-associated virus-mediated cancer gene therapy: current status. Cancer Lett. 2015;356(2 Pt B):347–356. doi: 10.1016/j.canlet.2014.10.045.

9. Santiago-Ortiz J.L., Schaffer D.V. Adeno-associated virus (AAV) vectors in cancer gene therapy. J. Control Release. 2016;240:287–301. doi: 10.1016/j.jconrel.2016.01.001.

10. Xue J., Chen K., Hu H., Gopinath S.C.B. Progress in gene therapy treatments for prostate cancer. Biotechnol. Appl. Biochem. 2022;69:1166–1175. doi: 10.1002/bab.2193.

11. Yahya E.B., Alqadhi A.M. Recent trends in cancer therapy: a review on the current state of gene delivery. Life Sci. 2021;269:119087. doi: 10.1016/j.lfs.2021.119087.

12. Gonçalves M.A., Pau M.G., de Vries A.A., Valerio D. Generation of a high-capacity hybrid vector: packaging of recombinant adenoassociated virus replicative intermediates in adenovirus capsids overcomes the limited cloning capacity of adenoassociated virus vectors. Virology. 2001;288:236–246. doi: 10.1006/viro.2001.1073.

13. Belver L., Ferrando A. The genetics and mechanisms of T cell acute lymphoblastic leukaemia. Nat. Rev. Cancer. 2016;16:494–507. doi: 10.1038/nrc.2016.63.

14. Bigas A., Rodriguez-Sevilla J.J., Espinosa L., Gallardo F. Recent advances in T-cell lymphoid neoplasms. Exp. Hematol. 2022;106:3–18. doi: 10.1016/j.exphem.2021.12.191.

15. Stanchina M., Soong D., Zheng-Lin B., Watts J.M., Taylor J. Advances in acute myeloid leukemia: recently approved therapies and drugs in development. Cancers. 2020;12:E3225. doi: 10.3390/cancers12113225.

16. Tsafa E., Bentayebi K., Topanurak S., Yata T., Przystal J., Fongmoon D., Hajji N., Waramit S., Suwan K., Hajitou A. Doxorubicin improves cancer cell targeting by filamentous phage gene delivery vectors. Int. J. Mol. Sci. 2020;21:E7867. doi: 10.3390/ijms21217867.

17. Wang Y., Huang F., Cai H., Wu Y., He G., Tan W.S. The efficacy of combination therapy using adeno-associated virus-TRAIL targeting to telomerase activity and cisplatin in a mice model of hepatocellular carcinoma. J. Cancer Res. Clin. Oncol. 2010;136:1827–1837. doi: 10.1007/s00432-010-0841-8.

18. Meador C.B., Hata A.N. Acquired resistance to targeted therapies in NSCLC: updates and evolving insights. Pharmacol. Ther. 2020;210:107522. doi: 10.1016/j.pharmthera.2020.107522.

19. Meador C.B., Sequist L.V., Piotrowska Z. Targeting EGFR exon 20 insertions in non-small cell lung cancer: recent advances and clinical updates. Cancer Discov. 2021;11:2145–2157. doi: 10.1158/2159-8290.CD-21-0226.

20. Zhong L., Li Y., Xiong L., Wang W., Wu M., Yuan T., Yang W., Tian C., Miao Z., Wang T., Yang S. Small molecules in targeted cancer therapy: advances, challenges, and future perspectives. Signal Transduct. Target. Ther. 2021;6:201. doi: 10.1038/s41392-021-00572-w.

21. Bejarano L., Jordāo M.J.C., Joyce J.A. Therapeutic targeting of the tumor microenvironment. Cancer Discov. 2021;11:933–959. doi: 10.1158/2159-8290.CD-20-1808.

22. Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin. Oncol. 2002;29(6 Suppl 16):15–18. doi: 10.1053/sonc.2002.37263.

23. Xie Y., Hicks M.J., Kaminsky S.M., Moore M.A.S., Crystal R.G., Rafii A. AAV-mediated persistent bevacizumab therapy suppresses tumor growth of ovarian cancer. Gynecol. Oncol. 2014;135:325–332. doi: 10.1016/j.ygyno.2014.07.105.

24. Li T., Kang G., Wang T., Huang H. Tumor angiogenesis and anti-angiogenic gene therapy for cancer. Oncol. Lett. 2018;16:687–702. doi: 10.3892/ol.2018.8733.

25. Stroncek D.F., Berger C., Cheever M.A., Childs R.W., Dudley M.E., Flynn P., Gattinoni L., Heath J.R., Kalos M., Marincola F.M., et al. New directions in cellular therapy of cancer: a summary of the summit on cellular therapy for cancer. J. Transl. Med. 2012;10:48. doi: 10.1186/1479-5876-10-48.

26. Chyuan I.T., Chu C.L., Hsu P.N. Targeting the tumor microenvironment for improving therapeutic effectiveness in cancer immunotherapy: focusing on immune checkpoint inhibitors and combination therapies. Cancers. 2021;1188 doi: 10.3390/cancers13061188.

27. Shiravand Y., Khodadadi F., Kashani S.M.A., Hosseini-Fard S.R., Hosseini S., Sadeghirad H., Ladwa R., O'Byrne K., Kulasinghe A. Immune checkpoint inhibitors in cancer therapy. Curr. Oncol. 2022;29:3044–3060. doi: 10.3390/curroncol29050247.

28. Cripe T.P., Hutzen B., Currier M.A., Chen C.Y., Glaspell A.M., Sullivan G.C., Hurley J.M., Deighen M.R., Venkataramany A.S., Mo X., et al. Leveraging gene therapy to achieve long-term continuous or controllable expression of biotherapeutics. Sci. Adv. 2022;8:eabm1890. doi: 10.1126/sciadv.abm1890.

29. Maresso K.C., Tsai K.Y., Brown P.H., Szabo E., Lippman S., Hawk E.T. Molecular cancer prevention: current status and future directions. CA. Cancer J. Clin. 2015;65:345–383. doi: 10.3322/caac.21287.

30. Ntanasis-Stathopoulos I., Terpos E., Dimopoulos M.A. Optimizing immunomodulatory drug with proteasome inhibitor combinations in newly diagnosed multiple myeloma. Cancer J. 2019;25:2–10. doi: 10.1097/PPO.0000000000000348.

31. Miller K.D., Nogueira L., Devasia T., Mariotto A.B., Yabroff K.R., Jemal A., Kramer J., Siegel R.L. Cancer Treatment and Survivorship Statistics. CA Cancer J. Clin. 2022;72:409–436. doi: 10.3322/caac.21731.

32. Lovly C.M., Dahlman K.B., Fohn L.E., Su Z., Dias-Santagata D., Hicks D.J., Hucks D., Berry E., Terry C., Duke M., et al. Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials. PLoS One. 2012;7:e35309. doi: 10.1371/journal.pone.0035309.

33. Zivanov C., Li X., Shah K.P., Estrada L., Cutrer W.B., Hooks M., Keedy V., Dahlman K.B. Precision cancer medicine: dynamic learning of cancer biology in a clinically meaningful context. Med. Sci. Educ. 2021;31:1029–1042. doi: 10.1007/s40670-021-01267-1.

34. Creelan B.C., Wang C., Teer J.K., Toloza E.M., Yao J., Kim S., Landin A.M., Mullinax J.E., Saller J.J., Saltos A.N., et al. Tumor-infiltrating lymphocyte treatment for anti-PD-1-resistant metastatic lung cancer: a phase 1 trial. Nat. Med. 2021;27:1410–1418. doi: 10.1038/s41591-021-01462-y.

35. Aung T.N., Shafi S., Wilmott J.S., Nourmohammadi S., Vathiotis I., Gavrielatou N., Fernandez A., Yaghoobi V., Sinnberg T., Amaral T., et al. Objective assessment of tumor infiltrating lymphocytes as a prognostic marker in melanoma using machine learning algorithms. EBioMedicine. 2022;82:104143. doi: 10.1016/j.ebiom.2022.104143.

36. Marin-Acevedo J.A., Kimbrough E.O., Manochakian R., Zhao Y., Lou Y. Immunotherapies targeting stimulatory pathways and beyond. J. Hematol. Oncol. 2021;14:78. doi: 10.1186/s13045-021-01085-3.

37. Molina-Arcas M., Samani A., Downward J. Drugging the undruggable: advances on RAS targeting in cancer. Genes (Basel) 2021;12:899. doi: 10.3390/genes12060899.

38. Iacobucci I., Kimura S., Mullighan C.G. Biologic and therapeutic implications of genomic alterations in acute lymphoblastic leukemia. J. Clin. Med. 2021;10:3792. doi: 10.3390/jcm10173792.

39. Gonzalez-Fierro A., Dueñas-González A. Drug repurposing for cancer therapy, easier said than done. Semin. Cancer Biol. 2021;68:123–131. doi: 10.1016/j.semcancer.2019.12.012.

40. Juarez M., Schcolnik-Cabrera A., Dueñas-Gonzalez A. The multitargeted drug ivermectin: from an antiparasitic agent to a repositioned cancer drug. Am. J. Cancer Res. 2018;8:317–331.

41. Schcolnik-Cabrera A., Chávez-Blanco A., Domínguez-Gómez G., Taja-Chayeb L., Morales-Barcenas R., Trejo-Becerril C., Perez-Cardenas E., Gonzalez-Fierro A., Dueñas-González A. Orlistat as a FASN inhibitor and multitargeted agent for cancer therapy. Expert Opin. Investig. Drugs. 2018;27:475–489. doi: 10.1080/13543784.2018.1471132.

42. Milone M.C., Fish J.D., Carpenito C., Carroll R.G., Binder G.K., Teachey D., Samanta M., Lakhal M., Gloss B., Danet-Desnoyers G., et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol. Ther. 2009;17:1453–1464. doi: 10.1038/mt.2009.83.

43. Glass E.B., Masjedi S., Dudzinski S.O., Wilson A.J., Duvall C.L., Yull F.E., Giorgio T.D. Optimizing mannose "click" conjugation to polymeric nanoparticles for targeted siRNA delivery to human and murine macrophages. ACS Omega. 2019;4:16756–16767. doi: 10.1021/acsomega.9b01465.

44. Song S., Lyle M.J., Noble-Vranish M.L., Min-Tran D.M., Harrang J., Xiao W., Unger E.C., Miao C.H. Ultrasound-mediated gene delivery of factor VIII plasmids for hemophilia A gene therapy in mice. Mol. Ther. Nucleic Acids. 2022;27:916–926. doi: 10.1016/j.omtn.2022.01.006.

45. Liu S.X., Xia Z.S., Zhong Y.Q. Gene therapy in pancreatic cancer. World J. Gastroenterol. 2014;20:13343–13368. doi: 10.3748/wjg.v20.i37.13343.

46. Daya S., Berns K.I. Gene therapy using adeno-associated virus vectors. Clin. Microbiol. Rev. 2008;21:583–593. doi: 10.1128/CMR.00008-08.

47. Kawalekar O.U., O'Connor R.S., Fraietta J.A., Guo L., McGettigan S.E., Posey A.D., Patel P.R., Guedan S., Scholler J., Keith B., et al. Distinct signaling of coreceptors regulates specific metabolism pathways and impacts memory development in CAR T cells. Immunity. 2016;44:712. doi: 10.1016/j.immuni.2016.02.023.

48. Zhao Z., Condomines M., van der Stegen S.J.C., Perna F., Kloss C.C., Gunset G., Plotkin J., Sadelain M. Structural design of engineered costimulation determines tumor rejection kinetics and persistence of CAR T cells. Cancer Cell. 2015;28:415–428. doi: 10.1016/j.ccell.2015.09.004.

49. Tai Y.T., Anderson K.C. Targeting B-cell maturation antigen in multiple myeloma. Immunotherapy. 2015;7:1187–1199. doi: 10.2217/imt.15.77.

50. Shimabukuro-Vornhagen A., Gödel P., Subklewe M., Stemmler H.J., Schlößer H.A., Schlaak M., Kochanek M., Böll B., von Bergwelt-Baildon M.S. Cytokine release syndrome. J. Immunother. Cancer. 2018;6:56. doi: 10.1186/s40425-018-0343-9.

51. Mao Y., Xu X. Cytokine release syndrome after treatment of anti-CD19 CAR-T therapy with IL-6 knocking down in patients with central nervous system B-cell acute lymphocytic leukemia. Ann. Clin. Lab. Sci. 2021;51:790–794.

52. Giavridis T., van der Stegen S.J.C., Eyquem J., Hamieh M., Piersigilli A., Sadelain M. CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade. Nat. Med. 2018;24:731–738. doi: 10.1038/s41591-018-0041-7.

53. Hewitt S.L., Bailey D., Zielinski J., Apte A., Musenge F., Karp R., Burke S., Garcon F., Mishra A., Gurumurthy S., et al. Intratumoral IL12 mRNA therapy promotes TH1 transformation of the tumor microenvironment. Clin. Cancer Res. 2020;26:6284–6298. doi: 10.1158/1078-0432.CCR-20-0472.

54. Kodama Y., Nakashima M., Nagahara T., Oyama N., Hashizume J., Nakagawa H., Harasawa H., Muro T., Kurosaki T., Yamashita C., et al. Development of a DNA vaccine for melanoma metastasis by inhalation based on an analysis of transgene expression characteristics of naked pDNA and a ternary complex in mouse lung tissues. Pharmaceutics. 2020;12:540. doi: 10.3390/pharmaceutics12060540.

55. Ruan Q., Xi L., Boye S.L., Han S., Chen Z.J., Hauswirth W.W., Lewin A.S., Boulton M.E., Law B.K., Jiang W.G., et al. Development of an anti-angiogenic therapeutic model combining scAAV2-delivered siRNAs and noninvasive photoacoustic imaging of tumor vasculature development. Cancer Lett. 2013;332:120–129. doi: 10.1016/j.canlet.2012.11.016.

56. Zhang C., Wang Q.T., Liu H., Zhang Z.Z., Huang W.L. Advancement and prospects of tumor gene therapy. Chin. J. Cancer. 2011;30:182–188. doi: 10.5732/cjc.010.10074.

57. Chen J., Liu X., Chen X., Guo Z., Liu J., Hao J., Zhang J. Real-time monitoring of miRNA function in pancreatic cell lines using recombinant AAV-based miRNA Asensors. PLoS One. 2013;8:e66315. doi: 10.1371/journal.pone.0066315.

58. Yin L., Keeler G.D., Zhang Y., Hoffman B.E., Ling C., Qing K., Srivastava A. AAV3-miRNA vectors for growth suppression of human hepatocellular carcinoma cells in vitro and human liver tumors in a murine xenograft model in vivo. Gene Ther. 2021;28:422–434. doi: 10.1038/s41434-020-0140-1.

59. Barresi V., Musmeci C., Rinaldi A., Condorelli D.F. Transcript-targeted therapy based on RNA interference and antisense oligonucleotides: current applications and novel molecular targets. Int. J. Mol. Sci. 2022;23:8875. doi: 10.3390/ijms23168875.

60. Le B.T., Raguraman P., Kosbar T.R., Fletcher S., Wilton S.D., Veedu R.N. Antisense oligonucleotides targeting angiogenic factors as potential cancer therapeutics. Mol. Ther. Nucleic Acids. 2019;14:142–157. doi: 10.1016/j.omtn.2018.11.007.

61. Montaño-Samaniego M., Bravo-Estupiñan D.M., Méndez-Guerrero O., Alarcón-Hernández E., Ibáñez-Hernández M. Strategies for targeting gene therapy in cancer cells with tumor-specific promoters. Front. Oncol. 2020;10:605380. doi: 10.3389/fonc.2020.605380.

62. Yang L., Gu X., Yu J., Ge S., Fan X. Oncolytic virotherapy: from bench to bedside. Front. Cell Dev. Biol. 2021;9:790150. doi: 10.3389/fcell.2021.790150.

63. Hoare J.I., Osmani B., O'Sullivan E.A., Browne A., Campbell N., Metcalf S., Nicolini F., Saxena J., Martin S.A., Lockley M. Carvedilol targets beta-arrestins to rewire innate immunity and improve oncolytic adenoviral therapy. Commun. Biol. 2022;5:106. doi: 10.1038/s42003-022-03041-4.

64. Inoue M., Kim M., Inoue T., Tait M., Byrne T., Nitschké M., Murer P., Cha H., Subramanian A., De Silva N., et al. Oncolytic vaccinia virus injected intravenously sensitizes pancreatic neuroendocrine tumors and metastases to immune checkpoint blockade. Mol. Ther. Oncolytics. 2022;24:299–318. doi: 10.1016/j.omto.2021.12.016.

65. Marchini A., Bonifati S., Scott E.M., Angelova A.L., Rommelaere J. Oncolytic parvoviruses: from basic virology to clinical applications. Virol. J. 2015;12:6. doi: 10.1186/s12985-014-0223-y.

66. Adeyemi R.O., Landry S., Davis M.E., Weitzman M.D., Pintel D.J. Parvovirus minute virus of mice induces a DNA damage response that facilitates viral replication. PLoS Pathog. 2010;6:e1001141. doi: 10.1371/journal.ppat.1001141.

67. Lacroix J., Leuchs B., Li J., Hristov G., Deubzer H.E., Kulozik A.E., Rommelaere J., Schlehofer J.R., Witt O. Parvovirus H1 selectively induces cytotoxic effects on human neuroblastoma cells. Int. J. Cancer. 2010;127:1230–1239. doi: 10.1002/ijc.25168.

68. Hristov G., Krämer M., Li J., El-Andaloussi N., Mora R., Daeffler L., Zentgraf H., Rommelaere J., Marchini A. Through its nonstructural protein NS1, parvovirus H-1 induces apoptosis via accumulation of reactive oxygen species. J. Virol. 2010;84:5909–5922. doi: 10.1128/JVI.01797-09.

69. Abschuetz A., Kehl T., Geibig R., Leuchs B., Rommelaere J., Régnier-Vigouroux A. Oncolytic murine autonomous parvovirus, a candidate vector for glioma gene therapy, is innocuous to normal and immunocompetent mouse glial cells. Cell Tissue Res. 2006;325:423–436. doi: 10.1007/s00441-006-0199-z.

70. Nüesch J.P.F., Lacroix J., Marchini A., Rommelaere J. Molecular pathways: rodent parvoviruses--mechanisms of oncolysis and prospects for clinical cancer treatment. Clin. Cancer Res. 2012;18:3516–3523. doi: 10.1158/1078-0432.CCR-11-2325.

71. Grekova S.P., Raykov Z., Zawatzky R., Rommelaere J., Koch U. Activation of a glioma-specific immune response by oncolytic parvovirus Minute Virus of Mice infection. Cancer Gene Ther. 2012;19:468–475. doi: 10.1038/cgt.2012.20.

72. Hartley A., Kavishwar G., Salvato I., Marchini A. A roadmap for the success of oncolytic parvovirus-based anticancer therapies. Annu. Rev. Virol. 2020;7:537–557. doi: 10.1146/annurev-virology-012220-023606.

73. Fuller-Carter P.I., Basiri H., Harvey A.R., Carvalho L.S. Focused update on AAV-based gene therapy clinical trials for inherited retinal degeneration. BioDrugs. 2020;34:763–781. doi: 10.1007/s40259-020-00453-8.

74. Aslesh T., Yokota T. Restoring SMN expression: an overview of the therapeutic developments for the treatment of spinal muscular atrophy. Cells. 2022;11:417. doi: 10.3390/cells11030417.

75. Roberts Z.J., Better M., Bot A., Roberts M.R., Ribas A. Axicabtagene ciloleucel, a first-in-class CAR T cell therapy for aggressive NHL. Leuk. Lymphoma. 2018;59:1785–1796. doi: 10.1080/10428194.2017.1387905.

76. South E., Cox E., Meader N., Woolacott N., Griffin S. Strimvelis((R)) for treating severe combined immunodeficiency caused by adenosine deaminase deficiency: an evidence review group perspective of a NICE highly specialised technology evaluation. Pharmacoecon. Open. 2019;3:151–161. doi: 10.1007/s41669-018-0102-3.

77. Ylä-Herttuala S. Glybera finally recommended for approval as the first gene therapy drug in the European Union. Mol. Ther. 2012;20:1831–1832. doi: 10.1038/mt.2012.194.

78. Salmon F., Grosios K., Petry H. Safety profile of recombinant adeno-associated viral vectors: focus on alipogene tiparvovec (Glybera(R)) Expert Rev. Clin. Pharmacol. 2014;7:53–65. doi: 10.1586/17512433.2014.852065.

79. Liu B.L., Robinson M., Han Z.Q., Branston R.H., English C., Reay P., McGrath Y., Thomas S.K., Thornton M., Bullock P., et al. ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties. Gene Ther. 2003;10:292–303. doi: 10.1038/sj.gt.3301885.

80. Langfield K.K., Walker H.J., Gregory L.C., Federspiel M.J. Manufacture of measles viruses. Methods Mol. Biol. 2011;737:345–366. doi: 10.1007/978-1-61779-095-9_14.

81. Ungerechts G., Bossow S., Leuchs B., Holm P.S., Rommelaere J., Coffey M., Coffin R., Bell J., Nettelbeck D.M. Moving oncolytic viruses into the clinic: clinical-grade production, purification, and characterization of diverse oncolytic viruses. Mol. Ther. Methods Clin. Dev. 2016;3:16018. doi: 10.1038/mtm.2016.18.

82. Engeland C.E., Ungerechts G. Measles virus as an oncolytic immunotherapy. Cancers (Basel) 2021;13:544. doi: 10.3390/cancers13030544.

83. Macedo N., Miller D.M., Haq R., Kaufman H.L. Clinical landscape of oncolytic virus research in 2020. J. Immunother. Cancer. 2020;8:e001486. doi: 10.1136/jitc-2020-001486.

84. Seth R.B., Sun L., Chen Z.J. Antiviral innate immunity pathways. Cell Res. 2006;16:141–147. doi: 10.1038/sj.cr.7310019.

85. Lang S.I., Giese N.A., Rommelaere J., Dinsart C., Cornelis J.J. Humoral immune responses against minute virus of mice vectors. J. Gene Med. 2006;8:1141–1150. doi: 10.1002/jgm.940.

86. Grimm D., Kay M.A. From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy. Curr. Gene Ther. 2003;3:281–304. doi: 10.2174/1566523034578285.

87. Xiao W., Chirmule N., Berta S.C., McCullough B., Gao G., Wilson J.M. Gene therapy vectors based on adeno-associated virus type 1. J. Virol. 1999;73:3994–4003. doi: 10.1128/JVI.73.5.3994-4003.1999.

88. Wang Q., Firrman J., Wu Z., Pokiniewski K.A., Valencia C.A., Wang H., Wei H., Zhuang Z., Liu L., Wunder S.L., et al. High-density recombinant adeno-associated viral particles are competent vectors for in vivo transduction. Hum. Gene Ther. 2016;27:971–981. doi: 10.1089/hum.2016.055.

89. Wu Z., Asokan A., Samulski R.J. Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol. Ther. 2006;14:316–327. doi: 10.1016/j.ymthe.2006.05.009.

90. Kwon I., Schaffer D.V. Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer. Pharm. Res. 2008;25:489–499. doi: 10.1007/s11095-007-9431-0.

91. Lam A.K., Frabutt D., Li L., Xiao W. Chemical modifications of the capsid for redirecting and improving the efficacy of adeno-associated virus vectors. Hum. Gene Ther. 2021;32:1433–1438. doi: 10.1089/hum.2021.124.

92. Smith J.K., Agbandje-McKenna M. Creating an arsenal of Adeno-associated virus (AAV) gene delivery stealth vehicles. PLoS Pathog. 2018;14:e1006929. doi: 10.1371/journal.ppat.1006929.

93. Cao M., You H., Hermonat P.L. The X gene of adeno-associated virus 2 (AAV2) is involved in viral DNA replication. PLoS One. 2014;9:e104596. doi: 10.1371/journal.pone.0104596.

94. Wilmott P., Lisowski L., Alexander I.E., Logan G.J. A user's guide to the inverted terminal repeats of adeno-associated virus. Hum. Gene Ther. Methods. 2019;30:206–213. doi: 10.1089/hgtb.2019.276.

95. Kuzmin D.A., Shutova M.V., Johnston N.R., Smith O.P., Fedorin V.V., Kukushkin Y.S., van der Loo J.C.M., Johnstone E.C. The clinical landscape for AAV gene therapies. Nat. Rev. Drug Discov. 2021;20:173–174. doi: 10.1038/d41573-021-00017-7.

96. Mingozzi F., Liu Y.L., Dobrzynski E., Kaufhold A., Liu J.H., Wang Y., Arruda V.R., High K.A., Herzog R.W. Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer. J. Clin. Invest. 2003;111:1347–1356. doi: 10.1172/JCI16887.

97. Monahan P.E., Négrier C., Tarantino M., Valentino L.A., Mingozzi F. Emerging immunogenicity and genotoxicity considerations of adeno-associated virus vector gene therapy for hemophilia. J. Clin. Med. 2021;10:2471. doi: 10.3390/jcm10112471.

98. Sun J., Hua B., Chen X., Samulski R.J., Li C. Gene delivery of activated factor VII using alternative adeno-associated virus serotype improves hemostasis in hemophiliac mice with FVIII inhibitors and adeno-associated virus neutralizing antibodies. Hum. Gene Ther. 2017;28:654–666. doi: 10.1089/hum.2017.016.

99. Qin W., Xu G., Tai P.W.L., Wang C., Luo L., Li C., Hu X., Xue J., Lu Y., Zhou Q., et al. Large-scale molecular epidemiological analysis of AAV in a cancer patient population. Oncogene. 2021;40:3060–3071. doi: 10.1038/s41388-021-01725-5.

100. Gil-Farina I., Fronza R., Kaeppel C., Lopez-Franco E., Ferreira V., D'Avola D., Benito A., Prieto J., Petry H., Gonzalez-Aseguinolaza G., Schmidt M. Recombinant AAV integration is not associated with hepatic genotoxicity in nonhuman primates and patients. Mol. Ther. 2016;24:1100–1105. doi: 10.1038/mt.2016.52.

101. Hu A., Ding M., Zhu J., Liu J.Q., Pan X., Ghoshal K., Bai X.F. Intra-tumoral delivery of IL-27 using adeno-associated virus stimulates anti-tumor immunity and enhances the efficacy of immunotherapy. Front. Cell Dev. Biol. 2020;8:210. doi: 10.3389/fcell.2020.00210.

102. Ru Q., Li W., Wang X., Zhang S., Chen L., Zhang Y., Ge Y., Zu Y., Liu Y., Zheng D. Preclinical study of rAAV2-sTRAIL: pharmaceutical efficacy, biodistribution and safety in animals. Cancer Gene Ther. 2017;24:251–258. doi: 10.1038/cgt.2017.12.

103. Volak A., LeRoy S.G., Natasan J.S., Park D.J., Cheah P.S., Maus A., Fitzpatrick Z., Hudry E., Pinkham K., Gandhi S., et al. Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous delivery. J. Neurooncol. 2018;139:293–305. doi: 10.1007/s11060-018-2889-2.

104. Bertolini T.B., Shirley J.L., Zolotukhin I., Li X., Kaisho T., Xiao W., Kumar S.R.P., Herzog R.W. Effect of CpG depletion of vector genome on CD8(+) T cell responses in AAV gene therapy. Front. Immunol. 2021;12:672449. doi: 10.3389/fimmu.2021.672449.

105. Butterfield J.S.S., Hege K.M., Herzog R.W., Kaczmarek R. A molecular revolution in the treatment of hemophilia. Mol. Ther. 2020;28:997–1015. doi: 10.1016/j.ymthe.2019.11.006.

106. Ertl H.C.J. Immunogenicity and toxicity of AAV gene therapy. Front. Immunol. 2022;13:975803. doi: 10.3389/fimmu.2022.975803.

107. Bartel M.A., Weinstein J.R., Schaffer D.V. Directed evolution of novel adeno-associated viruses for therapeutic gene delivery. Gene Ther. 2012;19:694–700. doi: 10.1038/gt.2012.20.

108. Schwartz M., Likhite S., Meyer K. Onasemnogene abeparvovec-xioi: a gene replacement strategy for the treatment of infants diagnosed with spinal muscular atrophy. Drugs Today (Barc) 2021;57:387–399. doi: 10.1358/dot.2021.57.6.3264117.

109. Sidonio R.F., Jr., Pipe S.W., Callaghan M.U., Valentino L.A., Monahan P.E., Croteau S.E. Discussing investigational AAV gene therapy with hemophilia patients: a guide. Blood Rev. 2021;47:100759. doi: 10.1016/j.blre.2020.100759.

110. Duan D., Yue Y., Engelhardt J.F. Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison. Mol. Ther. 2001;4:383–391. doi: 10.1006/mthe.2001.0456.

111. Shin J.H., Pan X., Hakim C.H., Yang H.T., Yue Y., Zhang K., Terjung R.L., Duan D. Microdystrophin ameliorates muscular dystrophy in the canine model of duchenne muscular dystrophy. Mol. Ther. 2013;21:750–757. doi: 10.1038/mt.2012.283.

112. He S.S., Shi H.S., Yin T., Li Y.X., Luo S.T., Wu Q.J., Lu L., Wei Y.Q., Yang L. AAV-mediated gene transfer of human pigment epithelium-derived factor inhibits Lewis lung carcinoma growth in mice. Oncol. Rep. 2012;27:1142–1148. doi: 10.3892/or.2012.1621.

113. Rama Ballesteros A.R., Hernández R., Perazzoli G., Cabeza L., Melguizo C., Vélez C., Prados J. Specific driving of the suicide E gene by the CEA promoter enhances the effects of paclitaxel in lung cancer. Cancer Gene Ther. 2020;27:657–668. doi: 10.1038/s41417-019-0137-3.

114. Teschendorf C., Emons B., Muzyczka N., Graeven U., Schmiegel W. Efficacy of recombinant adeno-associated viral vectors serotypes 1, 2, and 5 for the transduction of pancreatic and colon carcinoma cells. Anticancer Res. 2010;30:1931–1935.

115. Wang Y.G., Huang P.P., Zhang R., Ma B.Y., Zhou X.M., Sun Y.F. Targeting adeno-associated virus and adenoviral gene therapy for hepatocellular carcinoma. World J. Gastroenterol. 2016;22:326–337. doi: 10.3748/wjg.v22.i1.326.

116. Sayroo R., Nolasco D., Yin Z., Colon-Cortes Y., Pandya M., Ling C., Aslanidi G. Development of novel AAV serotype 6 based vectors with selective tropism for human cancer cells. Gene Ther. 2016;23:18–25. doi: 10.1038/gt.2015.89.

117. Trepel M., Körbelin J., Spies E., Heckmann M.B., Hunger A., Fehse B., Katus H.A., Kleinschmidt J.A., Müller O.J., Michelfelder S. Treatment of multifocal breast cancer by systemic delivery of dual-targeted adeno-associated viral vectors. Gene Ther. 2015;22:848. doi: 10.1038/gt.2015.76.

118. Su H., Lu R., Chang J.C., Kan Y.W. Tissue-specific expression of herpes simplex virus thymidine kinase gene delivered by adeno-associated virus inhibits the growth of human hepatocellular carcinoma in athymic mice. Proc. Natl. Acad. Sci. USA. 1997;94:13891–13896. doi: 10.1073/pnas.94.25.13891.

119. Su H., Chang J.C., Xu S.M., Kan Y.W. Selective killing of AFP-positive hepatocellular carcinoma cells by adeno-associated virus transfer of the herpes simplex virus thymidine kinase gene. Hum. Gene Ther. 1996;7:463–470. doi: 10.1089/hum.1996.7.4-463.

120. Meumann N., Schmithals C., Elenschneider L., Hansen T., Balakrishnan A., Hu Q., Hook S., Schmitz J., Bräsen J.H., Franke A.C., et al. Hepatocellular carcinoma is a natural target for adeno-associated virus (AAV) 2 vectors. Cancers (Basel) 2022;14:427. doi: 10.3390/cancers14020427.

121. Glushakova L.G., Lisankie M.J., Eruslanov E.B., Ojano-Dirain C., Zolotukhin I., Liu C., Srivastava A., Stacpoole P.W. AAV3-mediated transfer and expression of the pyruvate dehydrogenase E1 alpha subunit gene causes metabolic remodeling and apoptosis of human liver cancer cells. Mol. Genet. Metab. 2009;98:289–299. doi: 10.1016/j.ymgme.2009.05.010.

122. Crommentuijn M.H., Kantar R., Noske D.P., Vandertop W.P., Badr C.E., Würdinger T., Maguire C.A., Tannous B.A. Systemically administered AAV9-sTRAIL combats invasive glioblastoma in a patient-derived orthotopic xenograft model. Mol. Ther. Oncolytics. 2016;3:16017. doi: 10.1038/mto.2016.17.

123. Xia Y., He J., Zhang H., Wang H., Tetz G., Maguire C.A., Wang Y., Onuma A., Genkin D., Tetz V., et al. AAV-mediated gene transfer of DNase I in the liver of mice with colorectal cancer reduces liver metastasis and restores local innate and adaptive immune response. Mol. Oncol. 2020;14:2920–2935. doi: 10.1002/1878-0261.12787.

124. Lu L., Luo S.T., Shi H.S., Li M., Zhang H.L., He S.S., Liu Y., Pan Y., Yang L. AAV2-mediated gene transfer of VEGF-Trap with potent suppression of primary breast tumor growth and spontaneous pulmonary metastases by long-term expression. Oncol. Rep. 2012;28:1332–1338. doi: 10.3892/or.2012.1915.

125. Papadakis E.D., Nicklin S.A., Baker A.H., White S.J. Promoters and control elements: designing expression cassettes for gene therapy. Curr. Gene Ther. 2004;4:89–113. doi: 10.2174/1566523044578077.

126. Lin J., Lalani A.S., Harding T.C., Gonzalez M., Wu W.W., Luan B., Tu G.H., Koprivnikar K., VanRoey M.J., He Y., et al. Inhibition of lymphogenous metastasis using adeno-associated virus-mediated gene transfer of a soluble VEGFR-3 decoy receptor. Cancer Res. 2005;65:6901–6909. doi: 10.1158/0008-5472.CAN-05-0408.

127. Li H., Han X., Yang S., Wang Y., Dong Y., Tang T. FOXP1 drives osteosarcoma development by repressing P21 and RB transcription downstream of P53. Oncogene. 2021;40:2785–2802. doi: 10.1038/s41388-021-01742-4.

128. Fuster J.L., Bautista F., González B., Fernández J.M., Rives S., Dapena J.L., Leukemia Working Group of the Spanish Society of Pediatric Hematology and Oncology SEHOP Blinatumomab to improve the outcome of children with relapsed B-cell acute lymphoblastic leukemia. Clin. Transl. Oncol. 2021;23:1963–1966. doi: 10.1007/s12094-021-02590-0.

129. Lee S., Ahn H.J. Anti-EpCAM-conjugated adeno-associated virus serotype 2 for systemic delivery of EGFR shRNA: its retargeting and antitumor effects on OVCAR3 ovarian cancer in vivo. Acta Biomater. 2019;91:258–269. doi: 10.1016/j.actbio.2019.04.044.

130. Buenrostro D., Mulcrone P.L., Owens P., Sterling J.A. The bone microenvironment: a fertile soil for tumor growth. Curr. Osteoporos. Rep. 2016;14:151–158. doi: 10.1007/s11914-016-0315-2.

131. Hanahan D., Weinberg R.A. Hallmarks of cancer: the next generation. Cell. 2011;144:646–674. doi: 10.1016/j.cell.2011.02.013.

132. Macedo F., Ladeira K., Pinho F., Saraiva N., Bonito N., Pinto L., Goncalves F. Bone metastases: an overview. Oncol. Rev. 2017;11:321. doi: 10.4081/oncol.2017.321.

133. Madel M.B., Elefteriou F. Mechanisms supporting the use of beta-blockers for the management of breast cancer bone metastasis. Cancers (Basel) 2021;13:2887. doi: 10.3390/cancers13122887.

134. Sterling J.A., Edwards J.R., Martin T.J., Mundy G.R. Advances in the biology of bone metastasis: how the skeleton affects tumor behavior. Bone. 2011;48:6–15. doi: 10.1016/j.bone.2010.07.015.

135. Welch D.R., Hurst D.R. Defining the hallmarks of metastasis. Cancer Res. 2019;79:3011–3027. doi: 10.1158/0008-5472.CAN-19-0458.

136. Caswell D.R., Swanton C. The role of tumour heterogeneity and clonal cooperativity in metastasis, immune evasion and clinical outcome. BMC Med. 2017;15:133. doi: 10.1186/s12916-017-0900-y.

137. Yasmin-Karim S., King M.R., Messing E.M., Lee Y.F. E-selectin ligand-1 controls circulating prostate cancer cell rolling/adhesion and metastasis. Oncotarget. 2014;5:12097–12110. doi: 10.18632/oncotarget.2503.

138. Li N., Liu M., Cao X., Li W., Li Y., Zhao Z. Identification of differentially expressed genes using microarray analysis and COL6A1 induction of bone metastasis in non-small cell lung cancer. Oncol. Lett. 2021;22:693. doi: 10.3892/ol.2021.12954.

139. Blanco M.A., LeRoy G., Khan Z., Alečković M., Zee B.M., Garcia B.A., Kang Y. Global secretome analysis identifies novel mediators of bone metastasis. Cell Res. 2012;22:1339–1355. doi: 10.1038/cr.2012.89.

140. Yao Y., Fang Z.P., Chen H., Yue L., Min D.L., Tang L.N., Yu W.X., Kung H.F., Lin M.C., Shen Z. HGFK1 inhibits bone metastasis in breast cancer through the TAK1/p38 MAPK signaling pathway. Cancer Gene Ther. 2012;19:601–608. doi: 10.1038/cgt.2012.38.

141. Thaker P.H., Han L.Y., Kamat A.A., Arevalo J.M., Takahashi R., Lu C., Jennings N.B., Armaiz-Pena G., Bankson J.A., Ravoori M., et al. Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma. Nat. Med. 2006;12:939–944. doi: 10.1038/nm1447.

142. Roodman G.D. Genes associate with abnormal bone cell activity in bone metastasis. Cancer Metastasis Rev. 2012;31:569–578. doi: 10.1007/s10555-012-9372-x.

143. Okui T., Hiasa M., Ryumon S., Ono K., Kunisada Y., Ibaragi S., Sasaki A., Roodman G.D., White F.A., Yoneda T. The HMGB1/RAGE axis induces bone pain associated with colonization of 4T1 mouse breast cancer in bone. J. Bone Oncol. 2021;26:100330. doi: 10.1016/j.jbo.2020.100330.

144. Page J.M., Merkel A.R., Ruppender N.S., Guo R., Dadwal U.C., Cannonier S., Basu S., Guelcher S.A., Sterling J.A. Matrix rigidity regulates the transition of tumor cells to a bone-destructive phenotype through integrin beta3 and TGF-beta receptor type II. Biomaterials. 2015;64:33–44. doi: 10.1016/j.biomaterials.2015.06.026.

145. Dadwal U.C., Merkel A.R., Page J.M., Kwakwa K.A., Kessler M., Rhoades J.A. 3D bone morphology alters gene expression, motility, and drug responses in bone metastatic tumor cells. Int. J. Mol. Sci. 2020;21:E6913. doi: 10.3390/ijms21186913.

146. Dadwal U., Falank C., Fairfield H., Linehan S., Rosen C.J., Kaplan D.L., Sterling J., Reagan M.R. Tissue-engineered 3D cancer-in-bone modeling: silk and PUR protocols. Bonekey Rep. 2016;5:842. doi: 10.1038/bonekey.2016.75.

147. Mulcrone P.L., Campbell J.P., Clément-Demange L., Anbinder A.L., Merkel A.R., Brekken R.A., Sterling J.A., Elefteriou F. Skeletal colonization by breast cancer cells is stimulated by an osteoblast and beta2AR-dependent neo-angiogenic switch. J. Bone Miner. Res. 2017;32:1442–1454. doi: 10.1002/jbmr.3133.

148. Elefteriou F. Role of sympathetic nerves in the establishment of metastatic breast cancer cells in bone. J. Bone Oncol. 2016;5:132–134. doi: 10.1016/j.jbo.2016.03.003.

149. Zhang Y., Coleman M., Brekken R.A. Perspectives on hypoxia signaling in tumor stroma. Cancers (Basel) 2021;13:3070. doi: 10.3390/cancers13123070.

150. Chandra R., Karalis J.D., Liu C., Murimwa G.Z., Voth Park J., Heid C.A., Reznik S.I., Huang E., Minna J.D., Brekken R.A. The colorectal cancer tumor microenvironment and its impact on liver and lung metastasis. Cancers (Basel) 2021;13:6206. doi: 10.3390/cancers13246206.

151. Bhowmick N.A., Moses H.L. Tumor-stroma interactions. Curr. Opin. Genet. Dev. 2005;15:97–101. doi: 10.1016/j.gde.2004.12.003.

152. Sharma P., Allison J.P. Dissecting the mechanisms of immune checkpoint therapy. Nat. Rev. Immunol. 2020;20:75–76. doi: 10.1038/s41577-020-0275-8.

153. Horak C.E., Lee J.H., Marshall J.C., Shreeve S.M., Steeg P.S. Tumor-vascular interactions and tumor dormancy. APMIS. 2008;116:586–601. doi: 10.1111/j.1600-0463.2008.01213.x.

154. Ostapoff K.T., Awasthi N., Cenik B.K., Hinz S., Dredge K., Schwarz R.E., Brekken R.A. PG545, an angiogenesis and heparanase inhibitor, reduces primary tumor growth and metastasis in experimental pancreatic cancer. Mol. Cancer Ther. 2013;12:1190–1201. doi: 10.1158/1535-7163.MCT-12-1123.

155. Mastri M., Rosario S., Tracz A., Frink R.E., Brekken R.A., Ebos J.M.L. The challenges of modeling drug resistance to antiangiogenic therapy. Curr. Drug Targets. 2016;17:1747–1754. doi: 10.2174/1389450117666151209123544.

156. Hosein A.N., Brekken R.A., Maitra A. Pancreatic cancer stroma: an update on therapeutic targeting strategies. Nat. Rev. Gastroenterol. Hepatol. Aug 2020;17:487–505. doi: 10.1038/s41575-020-0300-1.

157. Guo J., Zeng H., Shi X., Han T., Liu Y., Liu Y., Liu C., Qu D., Chen Y. A CFH peptide-decorated liposomal oxymatrine inactivates cancer-associated fibroblasts of hepatocellular carcinoma through epithelial-mesenchymal transition reversion. J. Nanobiotechnology. 2022;20:114. doi: 10.1186/s12951-022-01311-1.

158. Elhag O.A.O., Hu X.J., Wen-Ying Z., Li X., Yuan Y.Z., Deng L.F., Liu D.L., Liu Y.L., Hui G. Reconstructed adeno-associated virus with the extracellular domain of murine PD-1 induces antitumor immunity. Asian Pac. J. Cancer Prev. 2012;13:4031–4036. doi: 10.7314/apjcp.2012.13.8.4031.

159. Tan Z., Chiu M.S., Yan C.W., Man K., Chen Z. Eliminating mesothelioma by AAV-vectored, PD1-based vaccination in the tumor microenvironment. Mol. Ther. Oncolytics. 2021;20:373–386. doi: 10.1016/j.omto.2021.01.010.

160. Daya S., Cortez N., Berns K.I. Adeno-associated virus site-specific integration is mediated by proteins of the nonhomologous end-joining pathway. J. Virol. 2009;83:11655–11664. doi: 10.1128/JVI.01040-09.

161. Arruda V.R., Schuettrumpf J., Herzog R.W., Nichols T.C., Robinson N., Lotfi Y., Mingozzi F., Xiao W., Couto L.B., High K.A. Safety and efficacy of factor IX gene transfer to skeletal muscle in murine and canine hemophilia B models by adeno-associated viral vector serotype 1. Blood. 2004;103:85–92. doi: 10.1182/blood-2003-05-1446.

162. Lu H., Chen L., Wang J., Huack B., Sarkar R., Zhou S., Xu R., Ding Q., Wang X., Wang H., Xiao W. Complete correction of hemophilia A with adeno-associated viral vectors containing a full-size expression cassette. Hum. Gene Ther. 2008;19:648–654. doi: 10.1089/hum.2007.0182.

163. Mulcrone P.L., Zhang J., Pride P.M., Lam A.K., Frabutt D.A., Ball-Kell S.M., Xiao W. Genomic designs of rAAVs contribute to pathological changes in the livers and spleens of mice. Article. Adv. Cell Gene Ther. 2022 doi: 10.1155/2022/6807904. 20226807904.

164. Burger C., Gorbatyuk O.S., Velardo M.J., Peden C.S., Williams P., Zolotukhin S., Reier P.J., Mandel R.J., Muzyczka N. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol. Ther. 2004;10:302–317. doi: 10.1016/j.ymthe.2004.05.024.

165. Zincarelli C., Soltys S., Rengo G., Rabinowitz J.E. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 2008;16:1073–1080. doi: 10.1038/mt.2008.76.

166. Gray S.J., Foti S.B., Schwartz J.W., Bachaboina L., Taylor-Blake B., Coleman J., Ehlers M.D., Zylka M.J., McCown T.J., Samulski R.J. Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Hum. Gene Ther. 2011;22:1143–1153. doi: 10.1089/hum.2010.245.

167. La Bella T., Imbeaud S., Peneau C., Mami I., Datta S., Bayard Q., Caruso S., Hirsch T.Z., Calderaro J., Morcrette G., et al. Adeno-associated virus in the liver: natural history and consequences in tumour development. Gut. 2020;69:737–747. doi: 10.1136/gutjnl-2019-318281.

168. Wang Q., Dong B., Firrman J., Roberts S., Moore A.R., Cao W., Diao Y., Kapranov P., Xu R., Xiao W. Efficient production of dual recombinant adeno-associated viral vectors for factor VIII delivery. Hum. Gene Ther. Methods. 2014;25:261–268. doi: 10.1089/hgtb.2014.093.

169. Zanolini D., Merlin S., Feola M., Ranaldo G., Amoruso A., Gaidano G., Zaffaroni M., Ferrero A., Brunelleschi S., Valente G., et al. Extrahepatic sources of factor VIII potentially contribute to the coagulation cascade correcting the bleeding phenotype of mice with hemophilia A. Haematologica. 2015;100:881–892. doi: 10.3324/haematol.2014.123117.

170. Wang Q., Dong B., Firrman J., Wu W., Roberts S., Moore A.R., Liu L.S., Chin M.P.S., Diao Y., Kost J., Xiao W. Evaluation of the biological differences of canine and human factor VIII in gene delivery: implications in human hemophilia treatment. Gene Ther. 2016;23:597–605. doi: 10.1038/gt.2016.34.

171. Robson T., Hirst D.G. Transcriptional targeting in cancer gene therapy. J. Biomed. Biotechnol. 2003;2003:110–137. doi: 10.1155/S1110724303209074.

172. Kota J., Chivukula R.R., O'Donnell K.A., Wentzel E.A., Montgomery C.L., Hwang H.W., Chang T.C., Vivekanandan P., Torbenson M., Clark K.R., et al. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell. 2009;137:1005–1017. doi: 10.1016/j.cell.2009.04.021.

173. Lv F., Qiu Y., Zhang Y., Liu S., Shi J., Liu Y., Zheng D. Adeno-associated virus-mediated anti-DR5 chimeric antibody expression suppresses human tumor growth in nude mice. Cancer Lett. 2011;302:119–127. doi: 10.1016/j.canlet.2011.01.001.

174. Di Scala M., Gil-Fariña I., Olagüe C., Vales A., Sobrevals L., Fortes P., Corbacho D., González-Aseguinolaza G. Identification of IFN-gamma-producing T cells as the main mediators of the side effects associated to mouse interleukin-15 sustained exposure. Oncotarget. 2016;7:49008–49026. doi: 10.18632/oncotarget.10264.

175. Jiang M., Liu Z., Xiang Y., Ma H., Liu S., Liu Y., Zheng D. Synergistic antitumor effect of AAV-mediated TRAIL expression combined with cisplatin on head and neck squamous cell carcinoma. BMC Cancer. 2011;11:54. doi: 10.1186/1471-2407-11-54.