Development of oncolytic and gene therapy vectors based on adenovirus serotype 4 as an alternative to adenovirus serotype 5.

BACKGROUND: Adenoviral vectors are among the most frequently used vectors for gene therapy and cancer treatment. Most vectors are derived from human adenovirus (Ad) serotype 5 despite limited applicability caused by pre-existing immunity and unfavorable liver tropism, whereas the other more than 100 known human serotypes remain largely unused. Here, we screened a library of human Ad types and identified Ad4 as a promising candidate vector. METHODS: Reporter-gene-expressing viruses representative of the natural human Ad diversity were used to transduce an array of muscle cell lines and two- or three-dimensional tumor cultures. The time-course of transgene expression was monitored by fluorescence or luminescence measurements. To generate replication-deficient Ad4 vector genomes, successive homologous recombination was applied. RESULTS: Ad4, 17 and 50 transduced human cardiomyocytes more efficiently than Ad5, whereas Ad37 was found to be superior in rhabdomyocytes. Despite its moderate transduction efficiency, Ad4 showed efficient and long-lasting gene expression in papillomavirus (HPV) positive tumor organoids. Therefore, we aimed to harness the potential of Ad4 for improved muscle transduction or oncolytic virotherapy of HPV-positive tumors. We deleted the E1 and E3 transcription units to produce first generation Ad vectors for gene therapy. The E1- and E1/E3-deleted vectors were replication-competent in HEK293 cells stably expressing E1 but not in the other cell lines tested. Furthermore, we show that the Ad5 E1 transcription unit can complement the replication of E1-deleted Ad4 vectors. CONCLUSIONS: Our Ad4-based gene therapy vector platform contributes to the development of improved Ad vectors based on non-canonical serotypes for a broad range of applications.

AAV11 enables efficient retrograde targeting of projection neurons and enhances astrocyte-directed transduction.

Viral tracers that enable efficient retrograde labeling of projection neurons are powerful vehicles for structural and functional dissections of the neural circuit and for the treatment of brain diseases. Currently, some recombinant adeno-associated viruses (rAAVs) based on capsid engineering are widely used for retrograde tracing, but display undesirable brain area selectivity due to inefficient retrograde transduction in certain neural connections. Here we developed an easily editable toolkit to produce high titer AAV11 and demonstrated that it exhibits potent and stringent retrograde labeling of projection neurons in adult male wild-type or Cre transgenic mice. AAV11 can function as a powerful retrograde viral tracer complementary to AAV2-retro in multiple neural connections. In combination with fiber photometry, AAV11 can be used to monitor neuronal activities in the functional network by retrograde delivering calcium-sensitive indicator under the control of a neuron-specific promoter or the Cre-lox system. Furthermore, we showed that GfaABC1D promoter embedding AAV11 is superior to AAV8 and AAV5 in astrocytic tropism in vivo, combined with bidirectional multi-vector axoastrocytic labeling, AAV11 can be used to study neuron-astrocyte connection. Finally, we showed that AAV11 allows for analyzing circuit connectivity difference in the brains of the Alzheimer's disease and control mice. These properties make AAV11 a promising tool for mapping and manipulating neural circuits and for gene therapy of some neurological and neurodegenerative disorders.

[Application prospect of conditional gene editing animals and viral vectors in experimental research of acupuncturology].

Conditional gene editing animals and viral vectors have been widely applied in the research fields of biology and medicine. Recently, they are also used as the effective approaches to reveal the underlying mechanism of acupuncture from the nervous system to the specific molecules. In order to further understand the application of conditional gene editing animals and viral vectors, in this article, we analyze their characteristics, advantages and recent development in the field of acupuncture research and discuss their potential roles and prospect in the future.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo.

OBJECTIVE: Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy. METHODS: The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression. RESULTS: rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity. CONCLUSION: Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

A Rapid and Efficient Vacuum-Based Agroinfiltration Protocol for Transient Gene Overexpression in Leaves of Catharanthus roseus.

Functional genomics analyses in planta can be hampered in non-model plants that are recalcitrant to the genetic transformation such as the medicinal plant Catharanthus roseus (Apocynaceae). No stable transformation and regeneration of plantlets have been achieved with a high efficiency in this plant to date. In addition, while virus-mediated transient gene silencing has been reported a decade ago in C. roseus, tools for transient overexpression remain scarce. Here, we describe an efficient and reliable methodology for transiently overexpressing any gene of interest in C. roseus leaves. This protocol combines a vacuum-based Agroinfiltration approach and the high translational efficiency of a deconstructed virus-based binary vector (pEAQ-HT). The described methodology is robust, easy to perform, and results in high amount of transient expression in C. roseus. This protocol is expected to serve as valuable tool to enhance the in planta characterization of gene functions or even transiently knock-in novel enzymatic activities.

Long-term functional correction of cystathionine ß-synthase deficiency in mice by adeno-associated viral gene therapy.

Cystathionine ß-synthase (CBS) deficiency is a recessive inborn error of sulfur metabolism characterized by elevated blood levels of total homocysteine (tHcy). Patients diagnosed with CBS deficiency are currently treated by a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine, but the effectiveness of this therapy is limited due to poor compliance. A mouse model for CBS deficiency (Tg-I278T Cbs-/- ) was used to evaluate a potential gene therapy approach to treat CBS deficiency utilizing an AAVrh.10-based vector containing the human CBS cDNA downstream of the constitutive, strong CAG promoter (AAVrh.10hCBS). Mice were administered a single dose of virus and followed for up to 1 year. The data demonstrated a dose-dependent increase in liver CBS activity and a dose-dependent decrease in serum tHcy. Liver CBS enzyme activity at 1 year was similar to Cbs+/- control mice. Mice given the highest dose (5.6 × 1011 genomes/mouse) had mean serum tHcy decrease of 97% 1 week after injection and an 81% reduction 1 year after injection. Treated mice had either full- or substantial correction of alopecia, bone loss, and fat mass phenotypes associated with Cbs deficiency in mice. Our findings show that AAVrh.10-based gene therapy is highly effective in treating CBS deficiency in mice and supports additional pre-clinical testing for eventual use human trials.

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy.

OBJECTIVE: Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells. METHODS: A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined. RESULTS: The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells. CONCLUSION: HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.

Oncolytic Virotherapy Treatment of Breast Cancer: Barriers and Recent Advances.

Oncolytic virotherapy (OV) is an emerging class of immunotherapeutic drugs. Their mechanism of action is two-fold: direct cell lysis and unmasking of the cancer through immunogenic cell death, which allows the immune system to recognize and eradicate tumours. Breast cancer is the most common cancer in women and is challenging to treat with immunotherapy modalities because it is classically an immunogenically "cold" tumour type. This provides an attractive niche for OV, given viruses have been shown to turn "cold" tumours "hot," thereby opening a plethora of treatment opportunities. There has been a number of pre-clinical attempts to explore the use of OV in breast cancer; however, these have not led to any meaningful clinical trials. This review considers both the potential and the barriers to OV in breast cancer, namely, the limitations of monotherapy and the scope for combination therapy, improving viral delivery and challenges specific to the breast cancer population (e.g., tumour subtype, menopausal status, age).

Development of Group B Coxsackievirus as an Oncolytic Virus: Opportunities and Challenges.

Oncolytic viruses have emerged as a promising strategy for cancer therapy due to their dual ability to selectively infect and lyse tumor cells and to induce systemic anti-tumor immunity. Among various candidate viruses, coxsackievirus group B (CVBs) have attracted increasing attention in recent years. CVBs are a group of small, non-enveloped, single-stranded, positive-sense RNA viruses, belonging to species human Enterovirus B in the genus Enterovirus of the family Picornaviridae. Preclinical studies have demonstrated potent anti-tumor activities for CVBs, particularly type 3, against multiple cancer types, including lung, breast, and colorectal cancer. Various approaches have been proposed or applied to enhance the safety and specificity of CVBs towards tumor cells and to further increase their anti-tumor efficacy. This review summarizes current knowledge and strategies for developing CVBs as oncolytic viruses for cancer virotherapy. The challenges arising from these studies and future prospects are also discussed in this review.

Synthetic Biology Medicine and Bacteria-Based Cancer Therapeutics.

Spontaneous tumor regression following bacterial infection has been observed for hundreds of years. These observations along with anecdotal medical findings in 1890s led to the development of Coley's "toxins," consisting of killed Streptococcus pyogenes and Serratia marcescens bacteria, as the first cancer immunotherapy. The use of this approach, however, was not widely accepted at the time especially after the introduction of radiation therapy as a treatment for cancer in the early 1900s. Over the last 30-40 years there has been renewed interest in the use of bacteria to treat human solid tumors. This is based on the observation that various nonpathogenic anaerobic bacteria can infiltrate and replicate within solid tumors when given intravenously. Bacteria tested as potential anticancer agents include the Gram-positive obligate anaerobes Bifidobacterium and Clostridium, as well as the gram-negative facultative anaerobe Salmonella. Recent advances in synthetic biology and clinical success in cancer immunotherapy provide renewed momentum for developing bacteria-based cancer immunotherapy for cancer treatment and should allow greater potential for the development of novel therapeutic approaches for this devastating disease.

Optimization of whole-brain rabies virus tracing technology for small cell populations.

The lateral hypothalamus (LH) is critically involved in the regulation of homeostatic energy balance. Some neurons in the LH express receptors for leptin (LepRb), a hormone known to increase energy expenditure and decrease energy intake. However, the neuroanatomical inputs to LepRb-expressing LH neurons remain unknown. We used rabies virus tracing technology to map these inputs, but encountered non-specific tracing. To optimize this technology for a minor cell population (LepRb is not ubiquitously expressed in LH), we used LepRb-Cre mice and assessed how different titers of the avian tumor virus receptor A (TVA) helper virus affected rabies tracing efficiency and specificity. We found that rabies expression is dependent on TVA receptor expression, and that leakiness of TVA receptors is dependent on the titer of TVA virus used. We concluded that a titer of 1.0-3.0 × 107 genomic copies per µl of the TVA virus is optimal for rabies tracing. Next, we successfully applied modified rabies virus tracing technology to map inputs to LepRb-expressing LH neurons. We discovered that other neurons in the LH itself, the periventricular hypothalamic nucleus (Pe), the posterior hypothalamic nucleus (PH), the bed nucleus of the stria terminalis (BNST), and the paraventricular hypothalamic nucleus (PVN) are the most prominent input areas to LepRb-expressing LH neurons.

Immunomodulation in Administration of rAAV: Preclinical and Clinical Adjuvant Pharmacotherapies.

Recombinant adeno-associated virus (rAAV) has attracted a significant research focus for delivering genetic therapies to target cells. This non-enveloped virus has been trialed in many clinical-stage therapeutic strategies but important obstacle in clinical translation is the activation of both innate and adaptive immune response to the protein capsid, vector genome and transgene product. In addition, the normal population has pre-existing neutralizing antibodies against wild-type AAV, and cross-reactivity is observed between different rAAV serotypes. While extent of response can be influenced by dosing, administration route and target organ(s), these pose concerns over reduction or complete loss of efficacy, options for re-administration, and other unwanted immunological sequalae such as local tissue damage. To reduce said immunological risks, patients are excluded if they harbor anti-AAV antibodies or have received gene therapy previously. Studies have incorporated immunomodulating or suppressive regimens to block cellular and humoral immune responses such as systemic corticosteroids pre- and post-administration of Luxturna® and Zolgensma®, the two rAAV products with licensed regulatory approval in Europe and the United States. In this review, we will introduce the current pharmacological strategies to immunosuppress or immunomodulate the host immune response to rAAV gene therapy.

Cloning and prokaryotic expression of the chicken liver kinase B1 (LKB1) and its localization in liver, heart and hypothalamus.

Liver kinase B1 (LKB1) is a member of the serine/threonine kinase family, which plays an indispensable role in the organism of animals. In the current study, the chicken LKB1 protein gene was amplified by PCR based on the primers and cDNA templates. Then, the cloning vector was constructed and the target gene was cloned. After that, the target gene was inserted into the expression vector to construct the recombinant plasmid. The recombinant plasmid was transformed into BL21 (DE3) host cells and the LKB1 recombinant proteins were successfully expressed by using Isopropyl-ß-D-thiogalactopyranoside (IPTG). Finally, purified LKB1 proteins were used as antigen and the rabbit-derived antiserums were collected. The antiserum titer determined by ELISA was not less than 1:128000. The results of Western blot suggested that the polyclonal antibody is highly specific to chicken LKB1 protein. Immunofluorescence indicated that the LKB1 protein is mainly expressed in the cytoplasm of liver, heart and hypothalamus cells of chicken. Our study showed that the LKB1 polyclonal antibodies produced by this method are effective and can be used to further study the role of LKB1 in the pathogenesis of chicken disease.

Rapid design and development of CRISPR-Cas13a targeting SARS-CoV-2 spike protein.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide epidemic of the lethal respiratory coronavirus disease (COVID-19), necessitating urgent development of specific and effective therapeutic tools. Among several therapeutic targets of coronaviruses, the spike protein is of great significance due to its key role in host invasion. Here, we report a potential anti-SARS-CoV-2 strategy based on the CRISPR-Cas13a system. Methods: A comprehensive set of bioinformatics methods, including sequence alignment, structural comparison, and molecular docking, was utilized to identify a SARS-CoV-2-spike(S)-specific segment. A tiling crRNA library targeting this specific RNA segment was designed, and optimal crRNA candidates were selected using in-silico methods. The efficiencies of the crRNA candidates were tested in human HepG2 and AT2 cells. Results: The most effective crRNA sequence inducing a robust cleavage effect on S and a potent collateral cleavage effect were identified. Conclusions: This study provides a rapid design pipeline for a CRISPR-Cas13a-based antiviral tool against SARS-CoV-2. Moreover, it offers a novel approach for anti-virus study even if the precise structures of viral proteins are indeterminate.

Gene therapy via canalostomy approach preserves auditory and vestibular functions in a mouse model of Jervell and Lange-Nielsen syndrome type 2.

Mutations in voltage-gated potassium channel KCNE1 cause Jervell and Lange-Nielsen syndrome type 2 (JLNS2), resulting in congenital deafness and vestibular dysfunction. We conducted gene therapy by injecting viral vectors using the canalostomy approach in Kcne1-/- mice to treat both the hearing and vestibular symptoms. Results showed early treatment prevented collapse of the Reissner's membrane and vestibular wall, retained the normal size of the semicircular canals, and prevented the degeneration of inner ear cells. In a dose-dependent manner, the treatment preserved auditory (16 out of 20 mice) and vestibular (20/20) functions in mice treated with the high-dosage for at least five months. In the low-dosage group, a subgroup of mice (13/20) showed improvements only in the vestibular functions. Results supported that highly efficient transduction is one of the key factors for achieving the efficacy and maintaining the long-term therapeutic effect. Secondary outcomes of treatment included improved birth and litter survival rates. Our results demonstrated that gene therapy via the canalostomy approach, which has been considered to be one of the more feasible delivery methods for human inner ear gene therapy, preserved auditory and vestibular functions in a dose-dependent manner in a mouse model of JLNS2.

Intracellular prodrug gene therapy for cancer mediated by tumor cell suicide gene exosomes.

Recently, we reported about exosomes possessing messenger RNA (mRNA) of suicide gene secreted from mesenchymal stem/stromal cells (MSCs) engineered to express the suicide gene-fused yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT). The yCD::UPRT-MSC exosomes are internalized by tumor cells and intracellularly convert prodrug 5-fluorocytosine (5-FC) to cytotoxic drug 5-fluorouracil (5-FU). Human tumor cells with the potential to metastasize release exosomes involved in the creation of a premetastatic niche at the predicted organs. We found that cancer cells stably transduced with yCD::UPRT gene by retrovirus infection released exosomes acting similarly like yCD::UPRT-MSC exosomes. Different types of tumor cells were transduced with the yCD::UPRT gene. The homogenous cell population of yCD::UPRT-transduced tumor cells expressed the yCD::UPRT suicide gene and secreted continuously exosomes with suicide gene mRNA in their cargo. All tumor cell suicide gene exosomes upon internalization into the recipient tumor cells induced the cell death by intracellular conversion of 5-FC to 5-FU and to 5-FUMP in a dose-dependent manner. Most of tumor cell-derived suicide gene exosomes were tumor tropic, in 5-FC presence they killed tumor cells but did not inhibit the growth of human skin fibroblast as well as DP-MSCs. Tumor cell-derived suicide gene exosomes home to their cells of origin and hold an exciting potential to become innovative specific therapy for tumors and potentially for metastases.

Early Phase Clinical Immunogenicity of Valoctocogene Roxaparvovec, an AAV5-Mediated Gene Therapy for Hemophilia A.

Evaluation of immune responses to adeno-associated virus (AAV)-mediated gene therapies prior to and following dose administration plays a key role in determining therapeutic safety and efficacy. This report describes up to 3 years of immunogenicity data following administration of valoctocogene roxaparvovec (BMN 270), an AAV5-mediated gene therapy encoding human B domain-deleted FVIII (hFVIII-SQ) in a phase 1/2 clinical study of adult males with severe hemophilia A. Patients with pre-existing humoral immunity to AAV5 or with a history of FVIII inhibitors were excluded from the trial. Blood plasma and peripheral blood mononuclear cell (PBMC) samples were collected at regular intervals following dose administration for assessment of humoral and cellular immune responses to both the AAV5 vector and transgene-expressed hFVIII-SQ. The predominant immune response elicited by BMN 270 administration was largely limited to the development of antibodies against the AAV5 capsid that were cross-reactive with other common AAV serotypes. No FVIII inhibitor responses were observed within 3 years following dose administration. In a context of prophylactic or on-demand corticosteroid immunosuppression given after vector infusion, AAV5 and hFVIII-SQ peptide-specific cellular immune responses were intermittently detected by an interferon (IFN)-γ and tumor necrosis factor (TNF)-α FluoroSpot assay, but they were not clearly associated with detrimental safety events or changes in efficacy measures.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic, or prophylactic. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin-converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in antiviral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH, and 6-O-desulfated enoxaparin with 50% inhibitory concentrations (IC50s) of 5.99 µg/liter, 1.08 mg/liter, 1.77 µg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes.IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.

Reversion mutation of cDNA CA8-204 minigene construct produces a truncated functional peptide that regulates calcium release in vitro and produces profound analgesia in vivo.

Intracellular calcium is critical in orchestrating neuronal excitability and analgesia. Carbonic anhydrase-8 (CA8) regulates intracellular calcium signaling through allosteric inhibition of neuronal inositol trisphosphate receptor 1 (ITPR1) to produce profound analgesia. Recently, we reported the "G" allele at rs6471859 represents cis-eQTL regulating alternative splicing of a 1697 bp transcript (CA8-204G) with a retained intron, alternative polyadenylation site and a new stop codon producing a functional 26 kDa peptide with an extended exon 3. In this study we show the reversion mutation (G to C) at rs6471859 within the CA8-204G expression vector also produced a stable 1697 bp transcript (CA8-204C) coding for a smaller peptide (~ 22 kDa) containing only the first three CA8 exons. Surprisingly, this peptide inhibited ITPR1 (pITPR1) activation, ITPR1-mediated calcium release in vitro; and produced profound analgesia in vivo. This is the first report showing CA8-204C codes for a functional peptide sufficient to regulate calcium signaling and produce profound analgesia.

Oncolytic Viruses as a Platform for the Treatment of Malignant Brain Tumors.

Malignant brain tumors remain incurable diseases. Although much effort has been devoted to improving patient outcome, multiple factors such as the high tumor heterogeneity, the strong tumor-induced immunosuppressive microenvironment, and the low mutational burden make the treatment of these tumors especially challenging. Thus, novel therapeutic strategies are urgent. Oncolytic viruses (OVs) are biotherapeutics that have been selected or engineered to infect and selectively kill cancer cells. Increasingly, preclinical and clinical studies demonstrate the ability of OVs to recruit T cells and induce durable immune responses against both virus and tumor, transforming a "cold" tumor microenvironment into a "hot" environment. Besides promising clinical results as a monotherapy, OVs can be powerfully combined with other cancer therapies, helping to overcome critical barriers through the creation of synergistic effects in the fight against brain cancer. Although many questions remain to be answered to fully exploit the therapeutic potential of OVs, oncolytic virotherapy will clearly be part of future treatments for patients with malignant brain tumors.