In vivo topical gene therapy for recessive dystrophic epidermolysis bullosa: a phase 1 and 2 trial.

Recessive dystrophic epidermolysis bullosa (RDEB) is a lifelong genodermatosis associated with blistering, wounding, and scarring caused by mutations in COL7A1, the gene encoding the anchoring fibril component, collagen VII (C7). Here, we evaluated beremagene geperpavec (B-VEC), an engineered, non-replicating COL7A1 containing herpes simplex virus type 1 (HSV-1) vector, to treat RDEB skin. B-VEC restored C7 expression in RDEB keratinocytes, fibroblasts, RDEB mice and human RDEB xenografts. Subsequently, a randomized, placebo-controlled, phase 1 and 2 clinical trial (NCT03536143) evaluated matched wounds from nine RDEB patients receiving topical B-VEC or placebo repeatedly over 12 weeks. No grade 2 or above B-VEC-related adverse events or vector shedding or tissue-bound skin immunoreactants were noted. HSV-1 and C7 antibodies sometimes presented at baseline or increased after B-VEC treatment without an apparent impact on safety or efficacy. Primary and secondary objectives of C7 expression, anchoring fibril assembly, wound surface area reduction, duration of wound closure, and time to wound closure following B-VEC treatment were met. A patient-reported pain-severity secondary outcome was not assessed given the small proportion of wounds treated. A global assessment secondary endpoint was not pursued due to redundancy with regard to other endpoints. These studies show that B-VEC is an easily administered, safely tolerated, topical molecular corrective therapy promoting wound healing in patients with RDEB.

Preclinical Evaluation of a Modified Herpes Simplex Virus Type 1 Vector Encoding Human TGM1 for the Treatment of Autosomal Recessive Congenital Ichthyosis.

Autosomal recessive congenital ichthyosis (ARCI) is a diverse group of cornification diseases associated with severe clinical complications and decreased quality of life. Germline mutations in the TGM1 gene, which encodes the enzyme TGM1, are the predominant cause of ARCI. These TGM1 mutations trigger the abnormal epidermal differentiation and impaired cutaneous barrier function observed in patients with ARCI. Unfortunately, current ARCI therapies focus solely on symptomatic relief. Thus, there is a significant unmet need for therapeutic strategies aimed at correcting the TGM1 deficiency underlying ARCI. In this study, we investigated the ability of KB105, a gene therapy vector encoding full-length human TGM1, to deliver functional human TGM1 to keratinocytes. In vitro, KB105 efficiently infected TGM1-deficient human keratinocytes, produced TGM1 protein, and rescued transglutaminase enzyme function. In vivo studies demonstrated that both single and repeated topical KB105 administration induced TGM1 protein expression in the target epidermal layer without triggering fibrosis, necrosis, or acute inflammation. Toxicity and biodistribution assessments on repeat dosing indicated that KB105 was well-tolerated and restricted to the dose site. Overall, our results demonstrate that rescuing TGM1 deficiency in patients with ARCI through topical KB105 application represents a promising strategy for safely and noninvasively treating this debilitating disease.

Phase 1 Study of Intravenous Oncolytic Poxvirus (vvDD) in Patients With Advanced Solid Cancers.

We have conducted a phase 1 study of intravenous vvDD, a Western Reserve strain oncolytic vaccinia virus, on 11 patients with standard treatment-refractory advanced colorectal or other solid cancers. The primary endpoints were maximum tolerated dose and associated toxicity while secondary endpoints were pharmacokinetics, pharmacodynamics, immune responses, and antitumor activity. No dose-limiting toxicities and treatment related severe adverse events were observed. The most common adverse events were grades 1/2 flu-like symptoms. Virus genomes were detectable in the blood 15-30 minutes after virus administration in a dose-dependent manner. There was evidence of a prolonged virus replication in tumor tissues in two patients, but no evidence of virus replication in non-tumor tissues, except a healed injury site and an oral thrush. Over 100-fold of anti-viral antibodies were induced in patients' sera. A strong induction of inflammatory and Th1, but not Th2 cytokines, suggested a potent Th1-mediated immunity against the virus and possibly the cancer. One patient showed a mixed response on PET-CT with resolution of some liver metastases, and another patient with cutaneous melanoma demonstrated clinical regression of some lesions. Given the confirmed safety, further trials evaluating intravenous vvDD in combination with therapeutic transgenes, immune checkpoint blockade or complement inhibitors, are warranted.

Complement Inhibition: A Novel Form of Immunotherapy for Colon Cancer.

BACKGROUND: Complement is a central part of both the innate and adaptive immune response and its activation has traditionally been considered part of the immunosurveillance response against cancer. Its pro-inflammatory role and its contribution to the development of many illnesses associated with inflammatory states implicate complement in carcinogenesis. METHODS: We evaluated the role of three protein inhibitors of complement-cobra venom factor, humanized cobra venom factor, and recombinant staphylococcus aureus superantigen-like protein 7-in the setting of a transplantable murine colon cancer model. Outcomes were evaluated by monitoring tumor growth, and flow cytometry, ELISPOT, and quantitative real-time PCR were used to determine the impact of complement inhibition on the host immune response. RESULTS: Complement inhibitors were effective at depleting complement component C3 in tumor bearing mice and this was temporally correlated with a decreased rate of tumor growth during the establishment of tumors. Treatment with cobra venom factor resulted in increased CD8(+) T cells as a percentage of tumor-infiltrating cells as well as a reduced immunosuppressive environment evidenced by decreased myeloid derived suppressor cells in splenocytes of treated mice. Complement inhibition resulted in increased expression of the chemoattractive cytokines CCL5, CXCL10, and CXCL11. DISCUSSION: Complement depletion represents a promising mode of immunotherapy in cancer by its ability to impair tumor growth by increasing the host's effective immune response to tumor and diminishing the immunosuppressive effect created by the tumor microenvironment and ultimately could be utilized as a component of combination immunotherapy.

First-in-man study of western reserve strain oncolytic vaccinia virus: safety, systemic spread, and antitumor activity.

Oncolytic viral therapy utilizes a tumor-selective replicating virus which preferentially infects and destroys cancer cells and triggers antitumor immunity. The Western Reserve strain of vaccinia virus (VV) is the most virulent strain of VV in animal models and has been engineered for tumor selectivity through two targeted gene deletions (vvDD). We performed the first-in-human phase 1, intratumoral dose escalation clinical trial of vvDD in 16 patients with advanced solid tumors. In addition to safety, we evaluated signs of vvDD replication and spread to distant tumors, pharmacokinetics and pharmacodynamics, clinical and immune responses to vvDD. Dose escalation proceeded without dose-limiting toxicities to a maximum feasible dose of 3 × 10(9) pfu. vvDD replication in tumors was reproducible. vvDD genomes and/or infectious particles were recovered from injected (n = 5 patients) and noninjected (n = 2 patients) tumors. At the two highest doses, vvDD genomes were detected acutely in blood in all patients while delayed re-emergence of vvDD genomes in blood was detected in two patients. Fifteen of 16 patients exhibited late symptoms, consistent with ongoing vvDD replication. In summary, intratumoral injection of the oncolytic vaccinia vvDD was well-tolerated in patients and resulted in selective infection of injected and noninjected tumors and antitumor activity.

A rationally designed A34R mutant oncolytic poxvirus: improved efficacy in peritoneal carcinomatosis.

Oncolytic poxviruses have demonstrated initial promising results in patients with cancer in clinical trials, yet further improvements are needed. It has been shown that a single point mutation in the A34R gene resulted in the production of more total progeny virus and more extracellular enveloped virus (EEV), a form that can be immune-evasive and with enhanced spread. We have genetically engineered a new oncolytic poxvirus (designated vA34R) by incorporating this mutated A34R gene into a viral backbone (vvDD) which was designed for tumor-selective replication. This rationally designed virus can evade neutralization from antipoxvirus antibodies and is highly cytotoxic to cancer cells. It demonstrates improved spread and increased replication within the peritoneal cavity resulting in improved antitumor effects in a peritoneal carcinomatosis (PC) model of MC38 colon cancer. Impressively, after carrier cell-mediated delivery in the preimmunized host, vA34R displayed high replication in tumor nodules yet low accumulation in normal tissues thus enhancing the therapeutic index leading to 70% long-term cures. These results demonstrate that vA34R gains an enhanced therapeutic index for PC via immune evasion, increased spread, and production of more progeny virus. Thus, vA34R may be a potent oncolytic virus (OV) for patients with PC, even after prior exposure to vaccinia virus (VV).

Mucin as a therapeutic target in pseudomyxoma peritonei.

Pseudomyxoma peritonei (PMP) is characterized by intraperitoneal dissemination of mucinous ascites. This malignancy frequently recurs despite aggressive locoregional therapies, demonstrates chemo-insensitivity and lacks targeted therapies. This review addresses some intriguing questions in PMP; what role does mucin play in this malignancy?; what genetic alterations and dysregulated signaling pathways lead to a putative goblet cell-lineage differentiation or mucin overexpression?; are targeted therapies against known transcriptional pathways for mucin production a novel therapeutic strategy in this malignancy?

Chronic anti-inflammatory drug therapy inhibits gel-forming mucin production in a murine xenograft model of human pseudomyxoma peritonei.

BACKGROUND: Intraperitoneal accumulation of mucinous ascites in pseudomyxoma peritonei (PMP) promotes an inflammatory/fibrotic reaction that progresses to bowel obstruction and eventual patient demise. Cytokines and inflammation-associated transcription factor binding sites, such as glucocorticoid response elements and COX-2, regulate secretory mucin, specifically MUC2, production. We hypothesized that anti-inflammatory drugs targeting inflammation-associated pathways may reduce mucin production and subsequent disease morbidity in PMP. METHODS: The effects of dexamethasone and Celebrex were assessed in mucin-secreting human colon cancer LS174T cells in vitro and murine xenograft models of LS174T and human appendiceal PMP in vivo by serial parametric measurements, MUC2 transcripts via real-time RT-PCR, and MUC2 protein expression via immunofluorescence assays. RESULTS: Dexamethasone significantly inhibited basal MUC2 mRNA levels in LS174T cells, inhibited mucinous tumor accumulation in an intraperitoneal PMP xenograft model, and prolonged survival in a subcutaneous LS174T xenograft model. Celebrex significantly inhibited sodium butyrate-stimulated MUC2 mRNA levels in LS174T cells and demonstrated a statistically nonsignificant trend toward reduced mucinous tumor growth and prolonged survival in the xenograft models. MUC2 protein analysis by immunofluorescence demonstrated a dual effect of dexamethasone on mucin production and tumor cell count. CONCLUSIONS: Inflammatory mediators are known to regulate mucin production and may promote overexpression of MUC2 by neoplastic cells with goblet cell phenotype in PMP. Anti-inflammatory drugs, dexamethasone and Celebrex, could inhibit extracellular mucin production in PMP by targeting inflammatory cascades and, therefore, may decrease compressive symptoms, increase the disease-free interval, and reduce the extent or frequency of morbid cytoreductive surgeries.

Expression of CCL19 from oncolytic vaccinia enhances immunotherapeutic potential while maintaining oncolytic activity.

Promising phase II clinical results have been reported recently for several oncolytic viral therapeutics, including strains based on vaccinia virus. One reason for this has been an increased appreciation of the critical therapeutic importance of the immune response raised by these viruses. However, the most commonly used approaches to enhance these immunotherapeutic effects in oncolytic viruses, typically though expression of cytokine transgenes, often also result in a reduction in oncolytic activity and premature clearance of the virotherapy from the tumor. Approaches that enhance the immunotherapeutic effects while maintaining oncolytic activity would therefore be beneficial. Here, it is demonstrated that the expression of the chemokine CCL19 (ELC) from an oncolytic vaccinia virus (vvCCL19) results in increased antitumor effects in syngeneic mouse tumor models. This corresponded with increased t cell and dendritic cell infiltration into the tumor. However, vvCCL19 persisted in the tumor at equivalent levels to a control virus without CCL19, demonstrating that oncolytic activity was not curtailed. Instead, vvCCL19 was cleared rapidly and selectively from normal tissues and organs, indicating a potentially increased safety profile. The therapeutic activity of vvCCL19 could be further significantly increased through combination with adoptive transfer of therapeutic immune cells expressing CCR7, the receptor for CCL19. This approach therefore represents a means to increase the safety and therapeutic benefit of oncolytic viruses, used alone or in combination with immune cell therapies.

Homeobox gene Rhox5 is regulated by epigenetic mechanisms in cancer and stem cells and promotes cancer growth.

BACKGROUND: Homeobox genes murine Rhox5 and human RHOXF1 are expressed in early embryonic stages and then mostly restricted to germline tissues in normal adult, yet they are aberrantly expressed in cancer cells in vitro and in vivo . Here we study the epigenetic regulation and potential functions of Rhox5 gene. FINDINGS: In Rhox5-silenced or extremely low expresser cells, we observed low levels of active histone epigenetic marks (H3ac, H4ac and H3K4me2) and high levels of repressive mark H3K9me2 along with DNA hypermethylation in the promoter. In Rhox5 low expresser cells, we typically observed modest levels of both active and repressive histone marks along with moderate DNA methylation. In Rhox5 highly expressed CT26 cancer cells, we observed DNA hypomethylation along with high levels of both active and repressive histone marks. Epigenetic drugs (retinoic acid and MS-275) induced F9 cell differentiation with enhanced Rhox5 expression and dynamic changes of epigenetic marks. Finally, Rhox5 knockdown by small hairpin RNA (shRNA) in CT26 colon cancer decreased cell proliferation and migration in vitro and tumor growth in vivo . CONCLUSIONS: Both DNA methylation and histone methylation/acetylation play key roles in modulating Rhox5 expression in various cell types. The stem cell-like "bivalent domain", an epigenetic feature originally identified in key differentiation genes within stem cells, exists in the Rhox5 gene promoter in not only embryonic stem cells but also cancer cells, cancer stem cells, and differentiated Sertoli cells. As Ras signaling-dependent Rhox5 expression promotes tumor growth, Rhox5 may be an ideal target for therapeutic intervention in cancer.

Chemokine expression from oncolytic vaccinia virus enhances vaccine therapies of cancer.

Tumor vaccines can induce robust immune responses targeting tumor antigens in the clinic, but antitumor effects have been disappointing. One reason for this is ineffective tumor infiltration of the cytotoxic T lymphocytes (CTLs) produced. Oncolytic viruses are capable of selectively replicating within tumor tissue and can induce a strong immune response. We therefore sought to determine whether these therapies could be rationally combined such that modulation of the tumor microenvironment by the viral therapy could help direct beneficial CTLs induced by the vaccine. As such, we examined the effects of expressing chemokines from oncolytic vaccinia virus, including CCL5 (RANTES), whose receptors are expressed on CTLs induced by different vaccines, including type-1-polarized dendritic cells (DC1). vvCCL5, an oncolytic vaccinia virus expressing CCL5, induced chemotaxis of lymphocyte populations in vitro and in vivo, and displayed improved safety in vivo. Interestingly, enhanced therapeutic benefits with vvCCL5 in vivo correlated with increased persistence of the viral agent exclusively within the tumor. When tumor-bearing mice were both vaccinated with DC1 and treated with vvCCL5 a further significant enhancement in tumor response was achieved which correlated with increased levels of tumor infiltrating lymphocytes. This approach therefore represents a novel means of combining biological therapies for cancer treatment.

Alternate mechanisms of initial pattern recognition drive differential immune responses to related poxviruses.

Vaccinia immunization was pivotal to successful smallpox eradication. However, the early immune responses that distinguish poxvirus immunization from pathogenic infection remain unknown. To address this, we developed a strategy to map the activation of key signaling networks in vivo and applied this approach to define and compare the earliest signaling events elicited by immunizing (vaccinia) and lethal (ectromelia) poxvirus infections in mice. Vaccinia induced rapid TLR2-dependent responses, leading to IL-6 production, which then initiated STAT3 signaling in dendritic and T cells. In contrast, ectromelia did not induce TLR2 activation, and profound mouse strain-dependent responses were observed. In resistant C57BL/6 mice, the STAT1 and STAT3 pathways were rapidly activated, whereas in susceptible BALB/c mice, IL-6-dependent STAT3 activation did not occur. These data link early immune signaling events to infection outcome and suggest that activation of different pattern-recognition receptors early after infection may be important in determining vaccine efficacy.

Establishment and characterization of a murine xenograft model of appendiceal mucinous adenocarcinoma.

We describe the clinical, pathologic and molecular characteristics of a xenograft model of metastatic mucinous appendiceal adenocarcinoma. Tumours from patients with mucinous appendiceal neoplasms were implanted in nude mice and observed for evidence of intraperitoneal tumour growth. Morphologic and immunohistochemical features, temporal growth characteristics relative to controls, and loss of heterozygosity (LOH) at multiple chromosomal alleles were assessed in a successfully engrafted tumour. Two of seventeen implanted tumours successfully engrafted and only one mucinous adenocarcinoma propagated throughout the course of the study. The successful xenograft is morphologically similar to the original tumour, produces abundant extracellular mucin and exhibits non-invasive growth on peritoneal surfaces. The temporal growth characteristics of the xenograft tumour relative to controls reveal that tumour burden can be followed indirectly by measuring the weight or abdominal girth of engrafted animals. The cytokeratin, mucin core protein, CDX2, Ki-67 and p53 expression patterns are identical in the xenograft and resected tumour and are consistent with the expected pattern of protein expression for mucinous adenocarcinoma of the appendix. LOH was found in 1 of 10 informative chromosomal loci (chromosome 10p23) in xenograft tumour cells. Although we were unable to engraft a low-grade appendiceal mucinous neoplasm, the engrafted adenocarcinoma will be useful for future evaluation of novel therapeutic strategies directed at mucinous appendiceal adenocarcinoma and evaluation of strategies for treating widespread, bulky, mucinous peritoneal surface neoplasms. Xenograft tumour enrichment can facilitate molecular studies of appendiceal epithelial neoplasia.

Three epigenetic drugs up-regulate homeobox gene Rhox5 in cancer cells through overlapping and distinct molecular mechanisms.

Epigenetic therapy of cancer using inhibitors of DNA methyltransferases (DNMT) or/and histone deacetylases (HDACs) has shown promising results in preclinical models and is being investigated in clinical trials. Homeodomain proteins play important roles in normal development and carcinogenesis. In this study, we demonstrated for the first time that an epigenetic drug could up-regulate homeobox genes in the reproductive homeobox genes on chromosome X (Rhox) family, including murine Rhox5, Rhox6, and Rhox9 and human RhoxF1 and RhoxF2 in breast, colon, and other types of cancer cells. We examined the molecular mechanisms underlining selective induction of Rhox5 in cancer cells by three epigenetic drugs: 5-aza-2'-deoxycytidine (DAC; decitabine), arsenic trioxide (ATO), and MS-275 [entinostat; N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide]. DAC induced Rhox5 mRNA expression from both distal promoter (Pd) and proximal promoter, whereas MS-275 and ATO induced gene expression from the Pd only. DAC and ATO inhibited both DNMT1 and DNMT3B protein expression, whereas MS-275 significantly reduced DNMT3B protein. In contrast to DAC, neither MS-275 nor ATO induced DNA demethylation on the Pd region. All three drugs led to enhanced acetylation of histones H3 and H4 at the promoter region. The occupancy of the activating histone mark dimethylated lysine 4 of H3 at Pd was enhanced by DAC and MS-275 but not ATO. Because they modulate gene expression with different potencies through shared and distinct epigenetic mechanisms, these epigenetic drugs may possess great potential in different applications for epigenetic therapy of cancer and other diseases.

The effect of elbow joint centre displacement on force generation and neural excitation.

Joint centre displacement may occur following total elbow replacement due to aseptic loosening or surgical misalignment, and has been linked to implant failure. In this study, the effects of joint centre displacement were examined using a neuromusculoskeletal model of the elbow joint. Isometric contractions were simulated at a range of joint angles during elbow flexion and extension. Displacement of the joint centre affected the force-generating capacity about the joint, due to changes in both muscle lengths and moment arms. The magnitude and direction of the maximum joint reaction force were also altered, potentially contributing to aseptic loosening and compromising joint stability. The relationship between force generated and the level of neural excitation to the elbow flexor and extensor muscles was also affected, suggesting that altered neural control patterns could be required following joint centre displacement.

Effect of elbow joint angle on force-EMG relationships in human elbow flexor and extensor muscles.

The purpose of this study was to examine the effect of joint angle on the relationship between force and electromyogram (EMG) amplitude and median frequency, in the biceps, brachioradialis and triceps muscles. Surface EMG were measured at eight elbow angles, during isometric flexion and extension at force levels from 10% to 100% of maximum voluntary contraction (MVC). Joint angle had a significant effect on MVC force, but not on MVC EMG amplitude in all of the muscles examined. The median frequency of the biceps and triceps EMG decreased with increasing muscle length, possibly due to relative changes in electrode position or a decrease in muscle fibre diameter. The relationship between EMG amplitude and force, normalised with respect to its maximum force at each angle, did not vary with joint angle in the biceps or brachioradialis muscles over all angles, or in the triceps between 45 degrees and 120 degrees of flexion. These results suggest that the neural excitation level to each muscle is determined by the required percentage of available force rather than the absolute force required. It is, therefore, recommended that when using surface EMG to estimate muscle excitation, force should be normalised with respect to its maximum value at each angle.

A neuromusculoskeletal model of the elbow joint for pre-clinical testing of total elbow replacement.

In this study, the effect of changing the geometry of the elbow joint was examined using a neuromusculoskeletal model. The position of the center of the joint was altered in order to simulate aseptic loosing or misalignment of the humeral component of a total elbow replacement. The effect of this change on model parameters, including the muscle moment arm and maximum wrist force, was examined. An isometric contraction with increasing voluntary drive was then simulated for a range of joint center positions, and the resulting joint reaction forces, and the direction of the force vectors were monitored. A change in the maximum force, measured at the wrist (17% - elbow flexion, 28% - extension), and in joint reaction force (up to 145N) was observed when the position of the joint center was altered. In addition, slight changes (up to 4.45 degrees ) in the direction of the joint reaction force vector were also observed.

Improved surface EMG electrode for measuring genioglossus muscle activity.

Activation of the genioglossus (GG) muscles is necessary to maintain the patency of the upper airway. In the condition of obstructive sleep apnea (OSA) this mechanism fails and the possible role of fatigue in its pathogenesis is still not fully understood. In this paper, a new electrode design for recording the genioglossus surface electromyogram (sEMG) is presented. The new design differs from a widely used GG surface electrode in both electrode configuration (unilateral rather than bilateral) and electrode material (Ag-AgCl rather than stainless steel (SS)). The separate effects of these factors were evaluated during force-varying and fatiguing contractions on normal human subjects and using GG sEMG model simulations. Unilateral sEMG was found to have lower amplitude, lower frequency content and a different rate of change of median frequency during fatiguing contractions. It was shown to overcome several disadvantages posed by the bilateral configuration and be more selective. Ag-AgCl has more favorable impedance characteristics and resulted in greater signal amplitudes. It was concluded that the new design is more suitable for detecting GG sEMG and allows more reliable interpretation of changes in sEMG due to physiological mechanisms, thus providing a new methodology for studying GG function and the role of fatigue in OSA.

Design of surface electrode array for measuring conduction velocity in the human genioglossus muscle.

A new appliance, incorporating linear arrays of pin electrodes for genioglossus (GG) surface electromyography measurement, is presented. This design enables the estimation of GG muscle fiber conduction velocity, which decreases with fatigue. The performance of the device was evaluated for ten healthy human subjects during fatiguing and force varying contractions.