Progerin induces a phenotypic switch in vascular smooth muscle cells and triggers replication stress and an aging-associated secretory signature.

Hutchinson-Gilford progeria syndrome is a premature aging disease caused by LMNA gene mutation and the production of a truncated prelamin A protein "progerin" that elicits cellular and organismal toxicity. Progerin accumulates in the vasculature, being especially detrimental for vascular smooth muscle cells (VSMC). Vessel stiffening and aortic atherosclerosis in HGPS patients are accompanied by VSMC depletion in the medial layer, altered extracellular matrix (ECM), and thickening of the adventitial layer. Mechanisms whereby progerin causes massive VSMC loss and vessel alterations remain poorly understood. Mature VSMC retain phenotypic plasticity and can switch to a synthetic/proliferative phenotype. Here, we show that progerin expression in human and mouse VSMC causes a switch towards the synthetic phenotype. This switch elicits some level of replication stress in normal cells, which is exacerbated in the presence of progerin, leading to telomere fragility, genomic instability, and ultimately VSMC death. Calcitriol prevents replication stress, telomere fragility, and genomic instability, reducing VSMC death. In addition, RNA-seq analysis shows induction of a profibrotic and pro-inflammatory aging-associated secretory phenotype upon progerin expression in human primary VSMC. Our data suggest that phenotypic switch-induced replication stress might be an underlying cause of VSMC loss in progeria, which together with loss of contractile features and gain of profibrotic and pro-inflammatory signatures contribute to vascular stiffness in HGPS.

Lamin A/C recruits ssDNA protective proteins RPA and RAD51 to stalled replication forks to maintain fork stability.

Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination-deficient cells, RFI in lamin A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin A/C. We suggest that RS is a major source of genomic instability in laminopathies and lamin A/C-deficient tumors.

T-Cell Mediated Inflammation in Postmenopausal Osteoporosis.

Osteoporosis is the most prevalent metabolic bone disease that affects half the women in the sixth and seventh decade of life. Osteoporosis is characterized by uncoupled bone resorption that leads to low bone mass, compromised microarchitecture and structural deterioration that increases the likelihood of fracture with minimal trauma, known as fragility fractures. Several factors contribute to osteoporosis in men and women. In women, menopause - the cessation of ovarian function, is one of the leading causes of primary osteoporosis. Over the past three decades there has been growing appreciation that the adaptive immune system plays a fundamental role in the development of postmenopausal osteoporosis, both in humans and in mouse models. In this review, we highlight recent data on the interactions between T cells and the skeletal system in the context of postmenopausal osteoporosis. Finally, we review recent studies on the interventions to ameliorate osteoporosis.

Ovariectomy Activates Chronic Low-Grade Inflammation Mediated by Memory T Cells, Which Promotes Osteoporosis in Mice.

The loss of estrogen (E2 ) initiates a rapid phase of bone loss leading to osteoporosis in one-half of postmenopausal women, but the mechanism is not fully understood. Here, we show for the first time how loss of E2 activates low-grade inflammation to promote the acute phase of bone catabolic activity in ovariectomized (OVX) mice. E2 regulates the abundance of dendritic cells (DCs) that express IL-7 and IL-15 by inducing the Fas ligand (FasL) and apoptosis of the DC. In the absence of E2 , DCs become long-lived, leading to increased IL-7 and IL-15. We find that IL-7 and IL-15 together, but not alone, induced antigen-independent production of IL-17A and TNFα in a subset of memory T cells (TMEM ). OVX of mice with T-cell-specific ablation of IL15RA showed no IL-17A and TNFα expression, and no increase in bone resorption or bone loss, confirming the role of IL-15 in activating the TMEM and the need for inflammation. Our results provide a new mechanism by which E2 regulates the immune system, and how menopause leads to osteoporosis. The low-grade inflammation is likely to cause or contribute to other comorbidities observed postmenopause. © 2020 American Society for Bone and Mineral Research.

Pulsed low-dose RANKL as a potential therapeutic for postmenopausal osteoporosis.

A number of studies in model animal systems and in the clinic have established that RANKL promotes bone resorption. Paradoxically, we found that pulsing ovariectomized mice with low-dose RANKL suppressed bone resorption, decreased the levels of proinflammatory effector T cells and led to increased bone mass. This effect of RANKL is mediated through the induction of FoxP3+CD25+ regulatory CD8+ T cells (TcREG) by osteoclasts. Here, we show that pulses of low-dose RANKL are needed to induce TcREG, as continuous infusion of identical doses of RANKL by pump did not induce TcREG. We also show that low-dose RANKL can induce TcREG at 2, 3, 6, and 10 weeks after ovariectomy. Our results show that low-dose RANKL treatment in ovariectomized mice is optimal at once-per-month doses to maintain the bone mass. Finally, we found that treatment of ovariectomized mice with the Cathepsin K inhibitor odanacatib also blocked TcREG induction by low-dose RANKL. We interpret this result to indicate that antigens presented to CD8+ T cells by osteoclasts are derived from the bone protein matrix because Cathepsin K degrades collagen in the bone. Taken together, our studies provide a basis for using low-dose RANKL as a potential therapeutic for postmenopausal osteoporosis.

Osteoclast-Primed Foxp3+ CD8 T Cells Induce T-bet, Eomesodermin, and IFN-γ To Regulate Bone Resorption.

Osteoimmunology arose from the recognition that cytokines produced by lymphocytes can affect bone homeostasis. We have previously shown that osteoclasts, cells that resorb bone, act as APCs. Cross-presentation of Ags by osteoclasts leads to expression of CD25 and Foxp3, markers of regulatory T cells in the CD8 T cells. Octeoclast-induced Foxp3(+) CD25(+) regulatory CD8 T cells (OC-iTcREG) suppress priming of CD4 and CD8 T cells by dendritic cells. OC-iTcREG also limit bone resorption by osteoclasts, forming a negative feedback loop. In this study, we show that OC-iTcREG express concurrently T-bet and Eomesodermin (Eomes) and IFN-γ. Pharmacological inhibition of IκK blocked IFN-γ, T-bet, and Eomes production by TcREG Furthermore, we show, using chromatin immunoprecipitation, NF-κB enrichment in the T-bet and Eomes promoters. We demonstrate that IFN-γ produced by TcREG is required for suppression of osteoclastogenesis and for degradation of TNFR-associated factor 6 in osteoclast precursors. The latter prevents signaling by receptor activator of NF-κB ligand needed for osteoclastogenesis. Knockout of IFN-γ rendered TcREG inefficient in preventing actin ring formation in osteoclasts, a process required for bone resorption. TcREG generated in vivo using IFN-γ(-/-) T cells had impaired ability to protect mice from bone resorption and bone loss in response to high-dose receptor activator of NF-κB ligand. The results of this study demonstrate a novel link between NF-κB signaling and induction of IFN-γ in TcREG and establish an important role for IFN-γ in TcREG-mediated protection from bone loss.

A Bone Anabolic Effect of RANKL in a Murine Model of Osteoporosis Mediated Through FoxP3+ CD8 T Cells.

TNF-α and IL-17 secreted by proinflammatory T cells (T(EFF)) promote bone erosion by activating osteoclasts. We previously demonstrated that in addition to bone resorption, osteoclasts act as antigen-presenting cells to induce FoxP3 in CD8 T cells (Tc(REG)). The osteoclast-induced regulatory CD8 T cells limit bone resorption in ovariectomized mice (a murine model of postmenopausal osteoporosis). Here we show that although low-dose receptor activator of NF-κB ligand (RANKL) maximally induces Tc(REG) via Notch signaling pathway to limit bone resorption, high-dose RANKL promotes bone resorption. In vitro, both TNF-α and IL-17, cytokines that are abundant in ovariectomized animals, suppress Tc(REG) induction by osteoclasts by repressing Notch ligand expression in osteoclasts, but this effect can be counteracted by addition of RANKL. Ovariectomized mice treated with low-dose RANKL induced Tc(REG) that suppressed bone resorption, decreased T(EFF) levels, and increased bone formation. High-dose RANKL had the expected osteolytic effect. Low-dose RANKL administration in ovariectomized mice lacking CD8 T cells was also osteolytic, confirming that Tc(REG) mediate this bone anabolic effect. Our results show that although RANKL directly stimulates osteoclasts to resorb bone, it also controls the osteoclasts' ability to induce regulatory T cells, engaging an important negative feedback loop. In addition to the conceivable clinical relevance to treatment of osteoporosis, these observations have potential relevance to induction of tolerance and autoimmune diseases.

Expanded anticancer therapeutic window of hexon-modified oncolytic adenovirus.

One of the significant hurdles toward safe and efficacious systemic treatment of cancer with oncolytic adenoviruses (Ads) is dose-limiting hepatotoxicity that prevents the increase of a therapeutic dose. In this study, we expanded the therapeutic window of oncolytic serotype 5 Ad (Ad5) by a genetic modification of hypervariable loop 5 (HVR5) in the capsid protein hexon that prevented infection of hepatocytes due to ablation of binding to blood factors. This oncolytic virus, Ad-GL-HB, had significantly reduced levels of hepatocyte transduction in immunocompetent and immunodeficient mice as compared to parental virus Ad-GL. The hepatocyte detargeting decreased liver damage and increased the maximum tolerated dose of Ad-GL-HB tenfold relative to that of Ad-GL. Intravenous (i.v.) injection of Ad-GL or Ad-GL-HB into tumor-bearing mice produced equally increased survival rates demonstrating that while Ad-GL-HB detargeted hepatocytes, it sustained tumor cell infection after systemic administration. The significantly improved safety of the virus allowed it to be used at increased doses for improved systemic antitumor efficacy. Our results suggest that hexon modifications provide valuable strategies for systemic oncolytic Ad therapy.

Characterization of human adenovirus serotypes 5, 6, 11, and 35 as anticancer agents.

Human adenovirus type 5 (Ad5) has been the most popular platform for the development of oncolytic Ads. Alternative Ad serotypes with low seroprevalence might allow for improved anticancer efficacy in Ad5-immune patients. We studied the safety and efficacy of rare serotypes Ad6, Ad11 and Ad35. In vitro cytotoxicity of the Ads correlated with expression of CAR and CD46 in most but not all cell lines. Among CAR-binding viruses, Ad5 was often more active than Ad6, among CD46-binding viruses Ad35 was generally more cytotoxic than Ad11 in cell culture studies. Ad5, Ad6, and Ad11 demonstrated similar anticancer activity in vivo, whereas Ad35 was not efficacious. Hepatotoxicity developed only in Ad5-injected mice. Predosing with Ad11 and Ad35 did not increase infection of hepatocytes with Ad5-based vector demonstrating different interaction of these Ads with Kupffer cells. Data obtained in this study suggest developing Ad6 and Ad11 as alternative Ads for anticancer treatment.

Systemic delivery of therapeutic viruses.

The treatment of certain diseases will require the systemic delivery of therapeutic genes or viruses. In most cases, intravascular injection is the best delivery method to achieve the systemic distribution of viruses and to enable these agents to reach distant therapeutic sites. However, viruses administered by intravascular injection encounter overlapping barriers that impede their ability to reach their targets, including interactions with blood cells, blood factors and endothelial cells, loss to hepatocytes and macrophages, and destruction by innate and adaptive immune responses. In this review, recent advances in the understanding of the mechanisms determining virus tropism following systemic administration and the pharmacology of therapeutic viruses are described. Adenoviruses are used as a paradigm of these interactions, and factors affecting their therapeutic efficacy and side effects are discussed, as well as how the barriers that impede their ability to reach their targets translate to other therapeutic viruses.

Chemical modification with high molecular weight polyethylene glycol reduces transduction of hepatocytes and increases efficacy of intravenously delivered oncolytic adenovirus.

Oncolytic adenoviruses are anticancer agents that replicate within tumors and spread to uninfected tumor cells, amplifying the anticancer effect of initial transduction. We tested whether coating the viral particle with polyethylene glycol (PEG) could reduce transduction of hepatocytes and hepatotoxicity after systemic (intravenous) administration of oncolytic adenovirus serotype 5 (Ad5). Conjugating Ad5 with high molecular weight 20-kDa PEG but not with 5-kDa PEG reduced hepatocyte transduction and hepatotoxicity after intravenous injection. PEGylation with 20-kDa PEG was as efficient at detargeting adenovirus from Kupffer cells and hepatocytes as virus predosing and warfarin. Bioluminescence imaging of virus distribution in two xenograft tumor models in nude mice demonstrated that PEGylation with 20-kDa PEG reduced liver infection 19- to 90-fold. Tumor transduction levels were similar for vectors PEGylated with 20-kDa PEG and unPEGylated vectors. Anticancer efficacy after a single intravenous injection was retained at the level of unmodified vector in large established prostate carcinoma xenografts, resulting in complete elimination of tumors in all animals and long-term tumor-free survival. Anticancer efficacy after a single intravenous injection was increased in large established hepatocellular carcinoma xenografts, resulting in significant prolongation of survival as compared with unmodified vector. The increase in efficacy was comparable to that obtained with predosing and warfarin pretreatment, significantly extending the median of survival. Shielding adenovirus with 20-kDa PEG may be a useful approach to improve the therapeutic window of oncolytic adenovirus after systemic delivery to primary and metastatic tumor sites.

Macrophage depletion combined with anticoagulant therapy increases therapeutic window of systemic treatment with oncolytic adenovirus.

Liver tropism of systemically delivered adenoviruses (Ad) represents a considerable challenge for their use as anticancer therapeutics. More than 90% of i.v. injected Ad is rapidly taken up by the liver leading to hepatotoxicity, reduced virus uptake by target tumor tissue, and diminished therapeutic efficacy. The lack of clinical activity of systemically given oncolytic Ad demands for better understanding and improvement of virus pharmacokinetics. We studied the effects of Ad "detargeting" from liver macrophages (Kupffer cells) and hepatocytes on toxicity and anticancer efficacy using a nonattenuated oncolytic Ad expressing enhanced green fluorescent protein-firefly luciferase fusion protein (Ad-EGFPLuc). Kupffer cell depletion before i.v. injection of Ad-EGFPLuc increased transgene expression in the liver 40.7-fold on day 3 after the injection indicating compensatory enhancement of hepatocyte transduction due to increased bioavailability of the virus. Pretreatment of mice with the anticoagulant drug warfarin to block blood factor-dependent binding of the virus to hepatocytes markedly reduced luciferase expression in the liver and mediated the corresponding decrease of hepatotoxicity in mice with intact and depleted liver macrophages. Combined depletion of Kupffer cells and pretreatment with warfarin before a single i.v. injection of Ad-EGFPLuc significantly reduced tumor growth and prolonged survival of nude mice bearing subcutaneous xenografts of aggressive human hepatocellular carcinoma. The improved antitumor activity correlated with enhanced transgene expression and virus spread in the tumors. These data suggest that detargeting oncolytic Ad from liver macrophages and hepatocytes is an effective strategy to increase the therapeutic window for therapy against disseminated tumor sites.

Reprogrammed viruses as cancer therapeutics: targeted, armed and shielded.

Virotherapy is currently undergoing a renaissance, based on our improved understanding of virus biology and genetics and our better knowledge of many different types of cancer. Viruses can be reprogrammed into oncolytic vectors by combining three types of modification: targeting, arming and shielding. Targeting introduces multiple layers of cancer specificity and improves safety and efficacy; arming occurs through the expression of prodrug convertases and cytokines; and coating with polymers and the sequential usage of different envelopes or capsids provides shielding from the host immune response. Virus-based therapeutics are beginning to find their place in cancer clinical practice, in combination with chemotherapy and radiation.

Modification of adenoviral vectors with polyethylene glycol modulates in vivo tissue tropism and gene expression.

Polyethylene glycol (PEG) is a hydrophilic polymer that has been used to coat adenoviral (Ad) vectors to improve their pharmacology. To analyze the effects of PEG on Ad5 tropism, Ad5 was covalently modified with different sizes of PEG and in vitro and in vivo transduction was analyzed. All tested PEGs ablated in vitro transduction. When protein C (PC) and factors VII, IX, and X were added, only factors IX and X increased transduction by the PEGylated vectors with the largest effect by X. Inactivation of these factors with warfarin drastically reduced liver transduction in mice by the PEGylated vectors after intravenous (i.v.) injection. Ad5 conjugated with 5 kd PEG maintained normal liver transduction while conjugation with larger 20 and 35 kd PEGs significantly reduced liver transduction. When intraperitoneal (i.p.) injection was tested, Ad transduced the peritoneum efficiently with only low level liver transduction. When Ad5 was modified with 5 kd PEG, peritoneal transduction was reduced and the virus preferentially transduced the liver. These data demonstrate the effects of different sizes of PEG on in vivo Ad tropism and suggest that this approach may be useful in retargeting and detargeting Ad in vivo.

Preparation and titration of CsCl-banded adenovirus stocks.

Adenovirus research often requires purified high-titer virus stocks and accurate virus titers for use in experiments. Accurate titers are important for quantitative, interpretable, and reproducible results. This is especially true when there are comparisons of different mutant viruses following infection. This chapter details the large-scale preparation of adenovirus (either replication-competent or replication-defective) in spinner cultures (e.g., KB, HeLa, or 293 cells). Protocols for harvesting cells and isolation of adenovirus by CsCl banding are presented. Methods for titering adenovirus by plaque assay are presented along with a discussion of how plaque assays can be used to determine the kinetics of cell killing and cytolysis by adenoviruses.

Targeting interferon-alpha increases antitumor efficacy and reduces hepatotoxicity of E1A-mutated spread-enhanced oncolytic adenovirus.

Novel approaches are needed to improve the antitumor potency and to increase the cancer specificity of oncolytic adenoviruses (Ad). We hypothesized that the combination of interferon-alpha (IFN-alpha) expression with a specific mutation in the e1a gene of Ad could target vector replication to genetic defects in the IFN-alpha pathway resulting in both improved antitumor efficacy and reduced toxicity. The conditionally replicative Ad vector KD3-IFN carries the dl1101/1107 mutation in the e1a gene that eliminates binding of E1A proteins to p300/CBP and pRb. KD3-IFN expresses human IFN-alpha in concurrence with vector replication and overexpresses the adenovirus death protein (ADP; E3-11.6K). The antitumor activity of KD3-IFN was significantly higher than that of a control vector in established human hepatocellular carcinoma tumors in immunodeficient mice and in hamster kidney cancer tumors in immunocompetent Syrian hamsters. The dl1101/1107 mutation rendered Ad replication sensitive to the antiviral effect of IFN-alpha in normal as opposed to cancer cells. These results translated to reduced vector toxicity upon systemic administration to C57BL/6 mice. The combination of Ad oncolysis, ADP overexpression, and IFN-alpha-mediated immunotherapy represents a three-pronged approach for increasing the anticancer efficacy of replicative Ads. Exploiting the dl1101/1107 mutation provides a mechanism for additional selectivity of IFN-alpha-expressing replication-competent Ads.

Targeting Interferon-α Increases Antitumor Efficacy and Reduces Hepatotoxicity of E1A-mutated Spread-enhanced Oncolytic Adenovirus.

Novel approaches are needed to improve the antitumor potency and to increase the cancer specificity of oncolytic adenoviruses (Ad). We hypothesized that the combination of interferon-alpha (IFN-α) expression with a specific mutation in the e1a gene of Ad could target vector replication to genetic defects in the IFN-α pathway resulting in both improved antitumor efficacy and reduced toxicity. The conditionally replicative Ad vector KD3-IFN carries the dl1101/1107 mutation in the e1a gene that eliminates binding of E1A proteins to p300/CBP and pRb. KD3-IFN expresses human IFN-α in concurrence with vector replication and overexpresses the adenovirus death protein (ADP; E3-11.6K). The antitumor activity of KD3-IFN was significantly higher than that of a control vector in established human hepatocellular carcinoma tumors in immunodeficient mice and in hamster kidney cancer tumors in immunocompetent Syrian hamsters. The dl1101/1107 mutation rendered Ad replication sensitive to the antiviral effect of IFN-α in normal as opposed to cancer cells. These results translated to reduced vector toxicity upon systemic administration to C57BL/6 mice. The combination of Ad oncolysis, ADP overexpression, and IFN-α-mediated immunotherapy represents a three-pronged approach for increasing the anticancer efficacy of replicative Ads. Exploiting the dl1101/1107 mutation provides a mechanism for additional selectivity of IFN-α-expressing replication-competent Ads.

Avian adenovirus vector CELO-TK displays anticancer activity in human cancer cells and suppresses established murine melanoma tumors.

Avian adenovirus CELO is a novel adenovirus vector system with the advantages of efficient production, high virion stability, and the absence of crossreactivity with Ad5-neutralizing antibodies. In this study, we evaluated the anticancer efficacy of a CELO vector encoding the herpes simplex virus type 1 thymidine kinase, a prodrug-activating therapeutic gene. Vectors carrying the gene for HSV-tk or EGFP under the control of the HCMV promoter in place of the "nonessential" region of the CELO genome were constructed. Anticancer activity of the CELO-TK vector was studied in vitro, in human and murine tumor cells in cell culture, and in vivo, in established subcutaneous murine B16 melanoma tumors in C57BL/6 mice. The CELO-TK vector mediated delivery of functional HSV-tk to tumor cell lines in cell culture. Comparison of the CELO-TK vector to a first-generation human adenovirus type 5 vector Ad5-TK in cultured H1299 cells showed equal levels of functional activity at increasing multiplicities of infection with CELO-based vector. CELO vectors allowed for transduction and expression of EGFP and HSV-tk genes in subcutaneous melanoma tumors in C57BL/6 mice. Intratumoral injections of CELO-TK followed by ganciclovir administration resulted in suppression of tumor growth and significantly increased the median of survival. The results of the study demonstrated the efficacy of CELO vector as a vehicle for the delivery of prodrug-activating genes such as HSV-tk to tumor cells in vitro and in vivo.