Efficacy of LCMV-based cancer immunotherapies is unleashed by intratumoral injections of polyI:C.

BACKGROUND: Lymphocytic choriomeningitis virus (LCMV) belongs to the Arenavirus family known for inducing strong cytotoxic T-cell responses in both mice and humans. LCMV has been engineered for the development of cancer immunotherapies, currently undergoing evaluation in phase I/II clinical trials. Initial findings have demonstrated safety and an exceptional ability to activate and expand tumor-specific T lymphocytes. Combination strategies to maximize the antitumor effectiveness of LCMV-based immunotherapies are being explored. METHODS: We assessed the antitumor therapeutic effects of intratumoral administration of polyinosinic:polycytidylic acid (poly(I:C)) and systemic vaccination using an LCMV-vector expressing non-oncogenic versions of the E6 and E7 antigens of human papillomavirus 16 (artLCMV-E7E6) in a bilateral model engrafting TC-1/A9 cells. This cell line, derived from the parental TC-1, exhibits low MHC class I expression and is highly immune-resistant. The mechanisms underlying the combination's efficacy were investigated through bulk RNA-seq, flow cytometry analyses of the tumor microenvironment, selective depletions using antibodies and clodronate liposomes, Batf3 deficient mice, and in vivo bioluminescence experiments. Finally, we assessed the antitumor effectiveness of the combination of artLCMV-E7E6 with BO-112, a GMP-grade poly(I:C) formulated in polyethyleneimine, currently under evaluation in clinical trials. RESULTS: Intratumoral injection of poly(I:C) enhanced the antitumor efficacy of artLCMV-E7E6 in both injected and non-injected tumor lesions. The combined treatment resulted in a significant delay in tumor growth and often complete eradication of several tumor lesions, leading to significantly improved survival compared with monotherapies. While intratumoral administration of poly(I:C) did not impact LCMV vector biodistribution or transgene expression, it significantly modified leucocyte infiltrates within the tumor microenvironment and amplified systemic efficacy through proinflammatory cytokines/chemokines such as CCL3, CCL5, CXCL10, TNF, IFNα, and IL12p70. Upregulation of MHC on tumor cells and a reconfiguration of the gene expression programs related to tumor vasculature, leucocyte migration, and the activation profile of tumor-infiltrating CD8+ T lymphocytes were observed. Indeed, the antitumor effect relied on the functions of CD8+ T lymphocytes and macrophages. The synergistic efficacy of the combination was further confirmed when BO-112 was included. CONCLUSION: Intratumoral injection of poly(I:C) sensitizes MHClow tumors to the antitumor effects of artLCMV-E7E6, resulting in a potent therapeutic synergy.

Peritoneal carcinomatosis in mouse models.

Peritoneal carcinomatosis (PCa) represents a metastatic stage of a disease with unmet therapeutic options. Malignant cells from primary tumors (gastrointestinal or gynecologic malignancies) invade the peritoneal cavity and eventually seed onto peritoneal surfaces, with the omentum being the most common nest area. With a median survival of less than 6 months, PCa has a dismal prognosis that can be improved with treatments only available to a select few individuals with low tumor burden. Thus, the discovery of novel and effective therapies for this disease depends on reliable animal models. Here, we describe a method to generate syngeneic PCa mouse models based on intraperitoneal (i.p.) administration of tumor cells. This model allows to follow-up cancer progression in PCa models from ovarian and colorectal origins monitoring mice bodyweight changes, ascites development and overall survival. Moreover, luciferase-expressing tumor cells can also be used to assess tumor growth after i.p. injection through in vivo bioluminescence quantification. The establishment of reliable, easy-to-monitor and reproducible intraperitoneal syngeneic tumors models, as described here, is the first step to develop cutting-edge therapies against PCa.

Intraperitoneal administration of a modified vaccinia virus Ankara confers single-chain interleukin-12 expression to the omentum and achieves immune-mediated efficacy against peritoneal carcinomatosis.

BACKGROUND: Peritoneal carcinomatosis is an advanced stage of cancer in which the disease has spread to the peritoneal cavity. In order to restore antitumor immunity subverted by tumor cells in this location, we evaluated intraperitoneal administrations of modified vaccinia virus Ankara (MVA) engineered to express single-chain interleukin 12 (scIL-12) to increase antitumor immune responses. METHODS: MVA encoding scIL-12 (MVA.scIL-12) was evaluated against peritoneal carcinomatosis models based on intraperitoneal engraftment of tumor cells. CD8-mediated immune responses, elucidated antitumor efficacy, and safety were evaluated following intravenous, intratumoral, or intraperitoneal administration of the viral vector. The immune response was measured by ELISpot (enzyme-linked immunosorbent spot), RNA sequencing, flow cytometry, intravital microscopy, and depletion of lymphocyte subsets with monoclonal antibodies. Safety was assessed by body-weight follow-up and blood testing. Tissue tropism on intravenous or intraperitoneal administration was assessed by bioluminescence analysis using a reporter MVA encoding luciferase. RESULTS: Intraperitoneal or locoregional administration, but not other routes of administration, resulted in a potent immune response characterized by increased levels of tumor-specific CD8+ T lymphocytes with the ability to produce both interferon-γ and tumor necrosis factor-α. The antitumor immune response was detectable not only in the peritoneal cavity but also systemically. As a result of intraperitoneal treatment, a single administration of MVA.scIL-12 encoding scIL-12 completely eradicated MC38 tumors implanted in the peritoneal cavity and also protected cured mice from subsequent subcutaneous rechallenges. Bioluminescence imaging using an MVA encoding luciferase revealed that intraperitoneal administration targets transgene to the omentum. The omentum is considered a key tissue in immune protection of the peritoneal cavity. The safety profile of intraperitoneal administration was also better than that following intravenous administration since no weight loss or hematological toxicity was observed when the vector was locally delivered into the peritoneal cavity. CONCLUSION: Intraperitoneal administration of MVA vectors encoding scIL-12 targets the omentum, which is the tissue where peritoneal carcinomatosis usually begins. MVA.scIL-12 induces a potent tumor-specific immune response that often leads to the eradication of experimental tumors disseminated to the peritoneal cavity.

Intratumoral injection of IL-12-encoding mRNA targeted to CSFR1 and PD-L1 exerts potent anti-tumor effects without substantial systemic exposure.

IL-12 is a potent cytokine for cancer immunotherapy. However, its systemic delivery as a recombinant protein has shown unacceptable toxicity in the clinic. Currently, the intratumoral injection of IL-12-encoding mRNA or DNA to avoid such side effects is being evaluated in clinical trials. In this study, we aimed to improve this strategy by further favoring IL-12 tethering to the tumor. We generated in vitro transcribed mRNAs encoding murine single-chain IL-12 fused to diabodies binding to CSF1R and/or PD-L1. These targeted molecules are expressed in the tumor microenvironment, especially on myeloid cells. The binding capacity of chimeric constructs and the bioactivity of IL-12 were demonstrated in vitro and in vivo. Doses as low as 0.5 µg IL-12-encoding mRNA achieved potent antitumor effects in subcutaneously injected B16-OVA and MC38 tumors. Treatment delivery was associated with increases in IL-12p70 and IFN-γ levels in circulation. Fusion of IL-12 to the diabodies exerted comparable efficacy against bilateral tumor models. However, it achieved tethering to myeloid cells infiltrating the tumor, resulting in nearly undetectable systemic levels of IL-12 and IFN-γ. Overall, tethering IL-12 to intratumoral myeloid cells in the mRNA-transferred tumors achieves similar efficacy while reducing the dangerous systemic bioavailability of IL-12.

Intratumoral immunotherapy with mRNAs encoding chimeric protein constructs encompassing IL-12, CD137 agonists, and TGF-ß antagonists.

Intratumoral immunotherapy strategies for cancer based on interleukin-12 (IL-12)-encoding cDNA and mRNA are under clinical development in combination with anti-PD-(L)1 monoclonal antibodies. To make the most of these approaches, we have constructed chimeric mRNAs encoding single-chain IL-12 fused to single-chain fragment variable (scFv) antibodies that bind to transforming growth factor ß (TGF-ß) and CD137 (4-1BB). Several neutralizing TGF-ß agents and CD137 agonists are also undergoing early-phase clinical trials. To attain TGF-ß and CD137 binding by the constructions, we used bispecific tandem scFv antibodies (taFvs) derived from the specific 1D11 and 1D8 monoclonal antibodies (mAbs), respectively. Transfection of mRNAs encoding the chimeric constructs achieved functional expression of the proteins able to act on their targets. Upon mRNA intratumoral injections in the transplantable mouse cancer models CT26, MC38, and B16OVA, potent therapeutic effects were observed following repeated injections into the tumors. Efficacy was dependent on the number of CD8+ T cells able to recognize tumor antigens that infiltrated the malignant tissue. Although the abscopal effects on concomitant uninjected lesions were modest, such distant effects on untreated lesions were markedly increased when combined with systemic PD-1 blockade.

Intracavitary adoptive transfer of IL-12 mRNA-engineered tumor-specific CD8+ T cells eradicates peritoneal metastases in mouse models.

Previous studies have shown that local delivery of tumor antigen-specific CD8+ T lymphocytes engineered to transiently express single-chain IL-12 mRNA is highly efficacious. Peritoneal dissemination of cancer is a frequent and often fatal patient condition usually diagnosed when the tumor burden is too large and hence uncontrollable with current treatment options. In this study, we have modeled intracavitary adoptive T cell therapy with OVA-specific OT-I T cells electroporated with IL-12 mRNA to treat B16-OVA and PANC02-OVA tumor spread in the peritoneal cavity. Tumor localization in the omentum and the effects of local T-cell encounter with the tumor antigens were monitored, the gene expression profile evaluated, and the phenotypic reprogramming of several immune subsets was characterized. Intraperitoneal administration of T cells promoted homing to the omentum more effectively than intravenous administration. Transient IL-12 expression was responsible for a favorable reprogramming of the tumor immune microenvironment, longer persistence of transferred T lymphocytes in vivo, and the development of immunity to endogenous antigens following primary tumor eradication. The efficacy of the strategy was at least in part recapitulated with the adoptive transfer of lower affinity transgenic TCR-bearing PMEL-1 T lymphocytes to treat the aggressive intraperitoneally disseminated B16-F10 tumor. Locoregional adoptive transfer of transiently IL-12-armored T cells appears to offer promising therapeutic advantages in terms of anti-tumor efficacy to treat peritoneal carcinomatosis.

Intratumoral Gene Transfer of mRNAs Encoding IL12 in Combination with Decoy-Resistant IL18 Improves Local and Systemic Antitumor Immunity.

IL12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs, and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding IL12 and IL18. Moreover, we have used a form of IL18 [decoy-resistant IL18 (DR-18)] which has preserved bioactivity but does not bind to the IL18 binding protein decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically cotransferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant noninjected lesions. Cotreatment was conducive to the presence of more activated CD8+ T cells in the treated and noninjected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL12 plus DR-18 local mRNA coinjection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

Omentum: Friend or foe in ovarian cancer immunotherapy?

Ovarian cancer often spreads out of the ovary before a patient is diagnosed and is the deadliest gynecological malignancy. The aggressiveness of ovarian cancer is determined by the progression in the form of peritoneal carcinomatosis, a stage with a poor prognosis and an untreatable condition in most patients. One of the first tumor nests or the origin of metastasis in the peritoneal cavity is the omentum. The omentum contains immune aggregates, called milky spots, embedded in adipose tissue, which support tumor growth by various mechanisms, including immunosuppressive immune cells and metabolic functions. In this sense, the abundance of blood vessels, omental resident macrophages, and chemokines, among other factors, are known to promote invasiveness, proliferation and resistance to cancer therapies. As a result, surgical practice employed in advanced-stage ovarian cancer almost constantly includes omentectomy. Paradoxically, the omentum is considered the "abdominal policeman" that contributes to peritoneal immunity by capturing antigens and pathogens from the peritoneal cavity and promoting effective immune responses against microbes. Why immunosurveillance against the metastatic tumor does not take place in the omentum? Could omental immune responses be activated with immunotherapeutic interventions? The omentum has largely been ignored in cancer immunology and immunotherapy, and the potential translational implications of this in ovarian cancer are still unclear. Here, we focus on the dual role of the omentum in ovarian cancer: its role in antitumor immune responses versus its activities fostering cancer progression.

Synergistic antitumor response with recombinant modified virus Ankara armed with CD40L and CD137L against peritoneal carcinomatosis.

Recombinant-modified vaccinia virus Ankara (rMVA) is known to elicit potent antitumor immune responses in preclinical models due to its inherent ability to activate the innate immune system and the activation of adaptive responses mediated by the expression of tumor antigens and costimulus-providing molecules, such as CD40L and CD137L. Here, we evaluated different rMVA vectors in preclinical peritoneal carcinomatosis models (ID8.OVA-Vegf/GFP and MC38). We compared rMVA vectors expressing a tumor antigen (OVA or gp70) either alone or co-expressed with CD40L or/and CD137L. In tumor-free mice, the vector coding for the triple combination was only slightly superior, whereas, in tumor-bearing animals, we observed a synergistic induction of T lymphocytes specific against vector-encoded and non-encoded tumor-associated antigens. The enhanced activation of the immune response was associated with improved survival in mice with peritoneal carcinomatosis treated with a rMVA vector encoding both CD40L and CD137L. Thus, the triple transgene combination in vaccinia viral vectors represents a promising strategy for the treatment of peritoneal carcinomatosis.

Overcoming the limitations of cytokines to improve cancer therapy.

Cytokines are pleiotropic soluble proteins used by immune cells to orchestrate a coordinated response against pathogens and malignancies. In cancer immunotherapy, cytokine-based drugs can be developed potentiating pro-inflammatory cytokines or blocking immunosuppressive cytokines. However, the complexity of the mechanisms of action of cytokines requires the use of biotechnological strategies to minimize systemic toxicity, while potentiating the antitumor response. Sequence mutagenesis, fusion proteins and gene therapy strategies are employed to enhance the half-life in circulation, target the desired bioactivity to the tumor microenvironment, and to optimize the therapeutic window of cytokines. In this review, we provide an overview of the different strategies currently being pursued in pre-clinical and clinical studies to make the most of cytokines for cancer immunotherapy.

Novel strategies exploiting interleukin-12 in cancer immunotherapy.

Interleukin-12 is considered a potent agent to enhance antitumor immune responses. It belongs to a family of heterodimeric cytokines with key roles in the up-regulation and down-regulation of cellular immunity. Since its discovery, recombinant IL-12 was found to exert potent antitumor effects in rodent tumor models and was rapidly tested in the clinic with an unfavorable benefit/toxicity profile. Localized delivery of IL-12 dramatically improves the therapeutic index and this approach is being applied in the clinic based on in-vivo electroporation of naked plasmid DNA encoding IL-12, mRNA formulations, viral vectors and tumor-targeted fusion proteins. Other biotechnology strategies such as IL-12-engineered local adoptive cell therapy and pro-cytokines can also be used to improve results and broaden the therapeutic window. Combination strategies of such localized IL-12-based approaches with checkpoint inhibitors are yielding promising results both preclinically and in the early-phase clinical trials.

Advances in mRNA-based drug discovery in cancer immunotherapy.

INTRODUCTION: Immune checkpoint inhibitors and adoptive T-cell therapy based on chimeric antigen receptors are the spearhead strategies to exploit the immune system to fight cancer. To take advantage of the full potential of the immune system, cancer immunotherapy must incorporate new biotechnologies such as mRNA technology that may synergize with already approved immunotherapies and act more effectively on immune targets. AREAS COVERED: This review describes the basics of mRNA biotechnology and provides insight into the recent advances in the use of mRNA for the local and systemic delivery of immunostimulatory antibodies, proinflammatory cytokines or for optimizing adoptive T-cell therapy. EXPERT OPINION: mRNA-based nanomedicines have great potential to expand the arsenal of immunotherapy tools due to their ability to simplify and accelerate drug development and their suitability for transient and local expression of immunostimulatory molecules, whose systemic and sustained expression would be toxic. The success of mRNA-based COVID-19 vaccines has highlighted the feasibility of this approach. Continuous advances in the delivery and construction of RNA-based vectors hold promise for improvements in clinical efficacy.

Statins act as transient type I interferon inhibitors to enable the antitumor activity of modified vaccinia Ankara viral vectors.

BACKGROUND: Modified vaccinia virus Ankara (MVA) are genetically engineered non-replicating viral vectors. Intratumoral administration of MVA induces a cyclic GMP-AMP synthase-mediated type I interferon (IFN) response and the production of high levels of the transgenes engineered into the viral genome such as tumor antigens to construct cancer vaccines. Although type I IFNs are essential for establishing CD8-mediated antitumor responses, this cytokine family may also give rise to immunosuppressive mechanisms. METHODS: In vitro assays were performed to evaluate the activity of simvastatin and atorvastatin on type I IFN signaling and on antigen presentation. Surface levels of IFN α/ß receptor 1, endocytosis of bovine serum albumin-fluorescein 5 (6)-isothiocyanate, signal transducer and activator of transcription (STAT) phosphorylation, and real-time PCR of IFN-stimulated genes were assessed in the murine fibroblast cell line L929. In vivo experiments were performed to characterize the effect of simvastatin on the MVA-induced innate immune response and on the antitumor effect of MVA-based antitumor vaccines in B16 melanoma expressing ovalbumin (OVA) and Lewis lung carcinoma (LLC)-OVA tumor models. RNAseq analysis, depleting monoclonal antibodies, and flow cytometry were used to evaluate the MVA-mediated immune response. RESULTS: In this work, we identified commonly prescribed statins as potent IFNα pharmacological inhibitors due to their ability to reduce surface expression levels of IFN-α/ß receptor 1 and to reduce clathrin-mediated endocytosis. Simvastatin and atorvastatin efficiently abrogated for 8 hours the transcriptomic response to IFNα and enhanced the number of dendritic cells presenting an OVA-derived peptide bound to major histocompatibility complex (MHC) class I. In vivo, intraperitoneal or intramuscular administration of simvastatin reduced the inflammatory response mediated by peritumoral administration of MVA and enhanced the antitumor activity of MVA encoding tumor-associated antigens. The synergistic antitumor effects critically depend on CD8+ cells, whereas they were markedly improved by depletion of CD4+ lymphocytes, T regulatory cells, or NK cells. Either MVA-OVA alone or combined with simvastatin augmented B cells, CD4+ lymphocytes, CD8+ lymphocytes, and tumor-specific CD8+ in the tumor-draining lymph nodes. However, only the treatment combination increased the numbers of these lymphocyte populations in the tumor microenvironment and in the spleen. CONCLUSION: In conclusion, blockade of IFNα functions by simvastatin markedly enhances lymphocyte infiltration and the antitumor activity of MVA, prompting a feasible drug repurposing.

Specific Protein 1 and p53 Interplay Modulates the Expression of the KCTD-Containing Cullin3 Adaptor Suppressor of Hedgehog 2.

The Hedgehog (Hh) signaling pathway plays a crucial role in normal embryonic development and adult tissue homeostasis. On the other end, dysregulated Hh signaling triggers a prolonged mitogenic response that may prompt abnormal cell proliferation, favoring tumorigenesis. Indeed, about 30% of medulloblastomas (MBs), the most common malignant childhood cerebellar tumors, exhibit improper activation of the Hh signaling. The oncosuppressor KCASH2 has been described as a suppressor of the Hh signaling pathway, and low KCASH2 expression was observed in Hh-dependent MB tumor. Therefore, the study of the modulation of KCASH2 expression may provide fundamental information for the development of new therapeutic approaches, aimed to restore physiological KCASH2 levels and Hh inhibition. To this end, we have analyzed the TATA-less KCASH2 proximal promoter and identified key transcriptional regulators of this gene: Sp1, a TF frequently overexpressed in tumors, and the tumor suppressor p53. Here, we show that in WT cells, Sp1 binds KCASH2 promoter on several putative binding sites, leading to increase in KCASH2 expression. On the other hand, p53 is involved in negative regulation of KCASH2. In this context, the balance between p53 and Sp1 expression, and the interplay between these two proteins determine whether Sp1 acts as an activator or a repressor of KCASH2 transcription. Indeed, in p53-/- MEF and p53 mutated tumor cells, we hypothesize that Sp1 drives promoter methylation through increased expression of the DNA methyltransferase 1 (DNMT1) and reduces KCASH2 transcription, which can be reversed by Sp1 inhibition or use of demethylating agents. We suggest therefore that downregulation of KCASH2 expression in tumors could be mediated by gain of Sp1 activity and epigenetic silencing events in cells where p53 functionality is lost. This work may open new venues for novel therapeutic multidrug approaches in the treatment of Hh-dependent tumors carrying p53 deficiency.

Mouse Models of Peritoneal Carcinomatosis to Develop Clinical Applications.

Peritoneal carcinomatosis of primary tumors originating in gastrointestinal (e.g., colorectal cancer, gastric cancer) or gynecologic (e.g., ovarian cancer) malignancies is a widespread type of tumor dissemination in the peritoneal cavity for which few therapeutic options are available. Therefore, reliable preclinical models are crucial for research and development of efficacious treatments for this condition. To date, a number of animal models have attempted to reproduce as accurately as possible the complexity of the tumor microenvironment of human peritoneal carcinomatosis. These include: Syngeneic tumor cell lines, human xenografts, patient-derived xenografts, genetically induced tumors, and 3D scaffold biomimetics. Each experimental model has its own strengths and limitations, all of which can influence the subsequent translational results concerning anticancer and immunomodulatory drugs under exploration. This review highlights the current status of peritoneal carcinomatosis mouse models for preclinical development of anticancer drugs or immunotherapeutic agents.

Interleukin-12 Message in a Bottle.

IL12 is a very potent cancer immunotherapy agent, but is difficult to harness safely if given systemically. Local gene transfer aims to confine the effects of IL12 to malignant tissues, thus avoiding toxicity. Lipid-nanoparticle mRNA achieves IL12 expression and efficacy in mouse models, opening the way to an ongoing trial.See related article by Hewitt et al., p. 6284.

Scavenger Receptor Class B Type I is Required for 25-Hydroxycholecalciferol Cellular Uptake and Signaling in Myeloid Cells.

SCOPE: Vitamin D3 is a critical molecule for the properly controlled activity of the immune system. In myeloid-derived cells, vitamin D3 induces the production of the antimicrobial and antitumor peptide cathelicidin. In this study, the mechanism of the entry of 25-hydroxycholecalciferol (25(OH)D) in myeloid-derived cells is explored. METHODS AND RESULTS: Here, a novel regulatory pathway of vitamin D3 biology is described. Using a polyclonal antibody, two different chemical inhibitors, and a high-density lipoprotein as a competing ligand, it is demonstrated here that the 25(OH)D signaling pathway in myeloid cells depends on scavenger receptor class B type I (SR-B1). This effect is observed in the THP-1 monocytic cell line and in human primary monocytes. SR-B1 blockade abrogates the cellular uptake of 25(OH)D leading to a general shut down of the gene transcription program modulated by 25(OH)D. The results obtained at the transcriptional level are confirmed at the protein and functional level for CD14 in the THP-1 cell line. CONCLUSION: In conclusion, SR-B1 plays a critical role in vitamin D3 biology, paving the way for novel therapeutic interventions.

Insulin Fused to Apolipoprotein A-I Reduces Body Weight and Steatosis in DB/DB Mice.

Background: Targeting long-lasting insulins to the liver may improve metabolic alterations that are not corrected with current insulin replacement therapies. However, insulin is only able to promote lipogenesis but not to block gluconeogenesis in the insulin-resistant liver, exacerbating liver steatosis associated with diabetes. Methods: In order to overcome this limitation, we fused a single-chain insulin to apolipoprotein A-I, and we evaluated the pharmacokinetics and pharmacodynamics of this novel fusion protein in wild type mice and in db/db mice using both recombinant proteins and recombinant adenoassociated virus (AAV). Results: Here, we report that the fusion protein between single-chain insulin and apolipoprotein A-I prolonged the insulin half-life in circulation, and accumulated in the liver. We analyzed the long-term effect of these insulin fused to apolipoprotein A-I or insulin fused to albumin using AAVs in the db/db mouse model of diabetes, obesity, and liver steatosis. While AAV encoding insulin fused to albumin exacerbated liver steatosis in several mice, AAV encoding insulin fused to apolipoprotein A-I reduced liver steatosis. These results were confirmed upon daily subcutaneous administration of the recombinant insulin-apolipoprotein A-I fusion protein for six weeks. The reduced liver steatosis was associated with reduced body weight in mice treated with insulin fused to apolipoprotein A-I. Recombinant apolipoprotein A-I alone significantly reduces body weight and liver weight, indicating that the apolipoprotein A-I moiety is the main driver of these effects. Conclusion: The fusion protein of insulin and apolipoprotein A-I could be a promising insulin derivative for the treatment of diabetic patients with associated fatty liver disease.

Long-Term Liver Expression of an Apolipoprotein A-I Mimetic Peptide Attenuates Interferon-Alpha-Induced Inflammation and Promotes Antiviral Activity.

Apolipoprotein A-I mimetic peptides are amphipathic alpha-helix peptides that display similar functions to apolipoprotein A-I. Preclinical and clinical studies have demonstrated the safety and efficacy of apolipoprotein A-I mimetic peptides in multiple indications associated with inflammatory processes. In this study, we evaluated the effect of the long-term expression of L37pA in the liver by an adeno-associated virus (AAV-L37pA) on the expression of an adeno-associated virus encoding interferon-alpha (AAV-IFNα). Long-term IFNα expression in the liver leads to lethal hematological toxicity one month after AAV administration. Concomitant administration of AAV-L37pA prevented the lethal toxicity since the IFNα expression was reduced one month after AAV administration. To identify the mechanism of action of L37pA, a genomic and proteomic analysis was performed 15 days after AAV administration when a similar level of IFNα and interferon-stimulated genes were observed in mice treated with AAV-IFNα alone and in mice treated with AAV-IFNα and AAV-L37pA. The coexpression of the apolipoprotein A-I mimetic peptide L37pA with IFNα modulated the gene expression program of IFNα, inducing a significant reduction in inflammatory pathways affecting pathogen-associated molecular patterns receptor, dendritic cells, NK cells and Th1 immune response. The proteomic analysis confirmed the impact of the L37pA activity on several inflammatory pathways and indicated an activation of LXR/RXR and PPPARα/γ nuclear receptors. Thus, long-term expression of L37pA induces an anti-inflammatory effect in the liver that allows silencing of IFNα expression mediated by an adeno-associated virus.

Treatment of Experimental Autoimmune Encephalomyelitis by Sustained Delivery of Low-Dose IFN-α.

Multiple sclerosis (MS) is a chronic autoimmune disease with no curative treatment. The immune regulatory properties of type I IFNs have led to the approval of IFN-ß for the treatment of relapsing-remitting MS. However, there is still an unmet need to improve the tolerability and efficacy of this therapy. In this work, we evaluated the sustained delivery of IFN-α1, either alone or fused to apolipoprotein A-1 by means of an adeno-associated viral (AAV) system in the mouse model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. These in vivo experiments demonstrated the prophylactic and therapeutic efficacy of the AAV-IFN-α or AAV-IFN-α fused to apolipoprotein A-1 vectors in experimental autoimmune encephalomyelitis, even at low doses devoid of hematological or neurologic toxicity. The sustained delivery of such low-dose IFN-α resulted in immunomodulatory effects, consisting of proinflammatory monocyte and T regulatory cell expansion. Moreover, encephalitogenic T lymphocytes from IFN-α-treated mice re-exposed to the myelin oligodendrocyte glycoprotein peptide in vitro showed a reduced proliferative response and cytokine (IL-17A and IFN-γ) production, in addition to upregulation of immunosuppressive molecules, such as IL-10, IDO, or PD-1. In conclusion, the results of the present work support the potential of sustained delivery of low-dose IFN-α for the treatment of MS and likely other T cell-dependent chronic autoimmune disorders.