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We have previously reported that an X-linked recessive form of chronic idiopathic intestinal pseudo-obstruction (CIIPX) maps to Xq28. To select candidate genes for the disease, we analyzed the expression in murine fetal brain and intestine of 56 genes from the critical region. We selected and sequenced seven genes and found that one affected male from a large CIIPX-affected kindred bears a 2-bp deletion in exon 2 of the FLNA gene that is present at the heterozygous state in the carrier females of the family. The frameshift mutation is located between two close methionines at the filamin N terminus and is predicted to produce a protein truncated shortly after the first predicted methionine. Loss-of-function FLNA mutations have been associated with X-linked dominant nodular ventricular heterotopia (PVNH), a central nervous system (CNS) migration defect that presents with seizures in females and lethality in males. Notably, the affected male bearing the FLNA deletion had signs of CNS involvement and potentially has PVNH. To understand how the severe frameshift mutation we found can explain the CIIPX phenotype and its X-linked recessive inheritance, we transiently expressed both the wild- type and mutant filamin in cell culture and found that filamin translation can start from either of the two initial methionines in these conditions. Therefore, translation of a normal shorter filamin can occur in vitro from the second methionine downstream of the 2-bp insertion we found. We confirmed this, demonstrating that the filamin protein is present in the patient's lymphoblastoid cell line that shows abnormal cytoskeletal actin organization compared with normal lymphoblasts. We conclude that the filamin N terminal region between the initial two methionines is crucial for proper enteric neuron development. [ABSTRACT FROM AUTHOR]

Oncolytic viruses (OVs) are novel anti-tumor agents with the ability to selectively infect and kill tumor cells while sparing normal tissue. Beyond tumor cytolysis, OVs are capable of priming an anti-tumor immune response via lysis and cross-presentation of locally expressed endogenous tumor antigens, acting as an "endovaccine." The effectiveness of OVs, similar to other immunotherapies, can be hampered by an immunosuppressive tumor microenvironment. In this study, we modified a previously generated oncolytic herpes simplex virus (oHSV) retargeted to the human HER2 (hHER2) tumor molecule and encoding murine interleukin-12 (mIL-12), by insertion of a second immunomodulatory molecule, murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), to maximize therapeutic efficacy. We assessed the efficacy of this double-armed virus (R-123) compared to singly expressing GM-CSF and IL-12 oHSVs in tumor-bearing mice. While monotherapies were poorly effective, combination with α-PD1 enhanced the anti-tumor response, with the highest efficacy of 100% response rate achieved by the combination of R-123 and α-PD1. Efficacy was T cell-dependent, and the induced immunity was long lasting and able to reject a second contralateral tumor. Importantly, systemic delivery of R-123 combined with α-PD1 was effective in inhibiting the development of tumor metastasis. As such, this approach could have a significant therapeutic impact paving the way for further development of this platform in cancer immunotherapy., (© 2020 The Author(s).)

Mucopolysaccharidosis VI (MPS VI) is caused by deficient arylsulfatase B (ARSB) activity resulting in lysosomal storage of glycosaminoglycans (GAGs). MPS VI is characterized by dysostosis multiplex, organomegaly, corneal clouding, and heart valve thickening. Gene transfer to a factory organ like liver may provide a lifetime source of secreted ARSB. We show that intravascular administration of adeno-associated viral vectors (AAV) 2/8-TBG-felineARSB in MPS VI cats resulted in ARSB expression up to 1 year, the last time point of the study. In newborn cats, normal circulating ARSB activity was achieved following delivery of high vector doses (6 × 10(13) genome copies (gc)/kg) whereas delivery of AAV2/8 vector doses as low as 2 × 10(12) gc/kg resulted in higher than normal serum ARSB levels in juvenile MPS VI cats. In MPS VI cats showing high serum ARSB levels, independent of the age at treatment, we observed: (i) clearance of GAG storage, (ii) improvement of long bone length, (iii) reduction of heart valve thickness, and (iv) improvement in spontaneous mobility. Thus, AAV2/ 8-mediated liver gene transfer represents a promising therapeutic strategy for MPS VI patients.

Mucopolysaccharidosis VI (MPS VI) is caused by deficient activity of arylsulfatase B (ARSB), resulting in intralysosomal storage of dermatan sulfate (DS) and multisystem disease without central nervous system involvement. After gene transfer, muscle or liver can theoretically be converted into factories for systemic ARSB secretion, leading to uptake by non-transduced cells. We have injected newborn MPS VI rats and cats with adeno-associated viral (AAV) vectors expressing ARSB under the control of liver-specific, muscle-specific, or universally active promoters. After systemic or intramuscular (IM) administration of AAV, therapeutic levels of circulating ARSB are achieved, resulting in skeletal improvements and significant decrease in glycosaminoglycan (GAG) storage, inflammation and apoptosis (despite a neutralizing immune response to ARSB in MPS VI rats). In addition, we have observed wide-spread dissemination of vector after IM AAV administration. This results in secretion of therapeutic levels of ARSB when the universally active cytomegalovirus (CMV) but not the muscle-specific muscle creatine kinase (MCK) promoter is used, suggesting that transduction of extramuscular sites rather than enzyme secretion from muscle occurs after muscle ARSB gene transfer. We conclude that AAV-mediated expression of ARSB from liver represents a feasible therapeutic strategy for MPS VI, potentially avoiding multiple infusions of costly recombinant enzyme associated with enzyme replacement therapy.

Ocular neovascularization associated with proliferative diabetic retinopathy and age-related macular degeneration is the leading cause of severe visual loss in adults in developed countries. Physiological and pathological retinal angiogenesis may occur independently in postnatal life through the complex activation of pro- and antiangiogenic pathways. We report that the Sonic hedgehog (Shh) pathway is activated in the retina in animal models of retinal and choroidal neovascularization. We show that pharmacological inhibition of the Shh signaling pathway significantly reduces physiological retinal angiogenesis and inhibits pathological vascularization in both models. Under retinal hypoxic conditions, inhibition of the Shh pathway results in reduction of vascular endothelial growth factor (VEGF) level, along with that of Patched-1 (Ptch1), a canonical Shh target, thus placing Shh activation upstream of VEGF in experimental retinal neovascularization. Our data demonstrate the requirement of the Shh pathway for retinal angiogenesis and its inhibition as a potential therapeutic strategy targeting ocular neovascular disease.

X-linked recessive ocular albinism type I (OA1) is due to mutations in the OA1 gene (approved gene symbol GPR143), which is expressed in the retinal pigment epithelium (RPE). The Oa1 (Gpr143) knockout mouse (Oa1(-/-)) model recapitulates many of the OA1 retinal morphological anomalies, including a lower number of melanosomes of increased size in the RPE. The Oa1(-/-) mouse also displays some of the retinal developmental abnormalities observed in albino patients such as misrouting of the optic tracts. Here, we show that these anomalies are associated with retinal electrophysiological abnormalities, including significant decrease in a- and b-wave amplitude and delayed recovery of b-wave amplitude from photoreceptor desensitization following bright light exposure. This suggests that lack of Oa1 in the RPE impacts on photoreceptor activity. More interestingly, adeno-associated viral vector-mediated Oa1 gene transfer to the retina of the Oa1(-/-) mouse model results in significant recovery of its retinal functional abnormalities. In addition, Oa1 retinal gene transfer increases the number of melanosomes in the Oa1(-/-) mouse RPE. Our data show that gene transfer to the adult retina unexpectedly rescues both functional and morphological abnormalities in a retinal developmental disorder, opening novel potential therapeutic perspectives for this and other forms of albinism.