Molecular therapy and nucleic acid adeno-associated virus-based gene therapy delivering combinations of two growth-associated genes to MPS IVA mice.

Mucopolysaccharidosis type IVA (MPS IVA) is caused by a deficiency of the galactosamine (N-acetyl)-6-sulfatase (GALNS) enzyme responsible for the degradation of specific glycosaminoglycans (GAGs). The progressive accumulation of GAGs leads to various skeletal abnormalities (short stature, hypoplasia, tracheal obstruction) and several symptoms in other organs. To date, no treatment is effective for patients with bone abnormalities. To improve bone pathology, we propose a novel combination treatment with the adeno-associated virus (AAV) vectors expressing GALNS enzyme and a natriuretic peptide C (CNP; NPPC gene) as a growth-promoting agent for MPS IVA. In this study, an MPS IVA mouse model was treated with an AAV vector expressing GALNS combined with another AAV vector expressing NPPC gene, followed for 12 weeks. After the combination therapy, bone growth in mice was induced with increased enzyme activity in tissues (bone, liver, heart, lung) and plasma. Moreover, there were significant changes in bone morphology in CNP-treated mice with increased CNP activity in plasma. Delivering combinations of CNP and GALNS gene therapies enhanced bone growth in MPS IVA mice more than in GALNS gene therapy alone. Enzyme expression therapy alone fails to reach the bone growth region; our results indicate that combining it with CNP offers a potential alternative.

Combination of T cell-redirecting strategies with a bispecific antibody blocking TGF-ß and PD-L1 enhances antitumor responses.

T cell-based immunotherapies for solid tumors have not achieved the clinical success observed in hematological malignancies, partially due to the immunosuppressive effect promoted by the tumor microenvironment, where PD-L1 and TGF-ß play a pivotal role. However, durable responses to immune checkpoint inhibitors remain limited to a minority of patients, while TGF-ß inhibitors have not reached the market yet. Here, we describe a bispecific antibody for dual blockade of PD-L1 and TFG-ß, termed AxF (scFv)2, under the premise that combination with T cell redirecting strategies would improve clinical benefit. The AxF (scFv)2 antibody was well expressed in mammalian and yeast cells, bound both targets and inhibited dose-dependently the corresponding signaling pathways in luminescence-based cellular reporter systems. Moreover, combined treatment with trispecific T-cell engagers (TriTE) or CAR-T cells significantly boosted T cell activation status and cytotoxic response in breast, lung and colorectal (CRC) cancer models. Importantly, the combination of an EpCAMxCD3×EGFR TriTE with the AxF (scFv)2 delayed CRC tumor growth in vivo and significantly enhanced survival compared to monotherapy with the trispecific antibody. In summary, we demonstrated the feasibility of concomitant blockade of PD-L1 and TGF-ß by a single molecule, as well as its therapeutic potential in combination with different T cell redirecting agents to overcome tumor microenvironment-mediated immunosuppression.

Iron oxide-coupled CRISPR-nCas9-based genome editing assessment in mucopolysaccharidosis IVA mice.

Mucopolysaccharidosis (MPS) IVA is a lysosomal storage disorder caused by mutations in the GALNS gene that leads to the lysosomal accumulation of keratan sulfate (KS) and chondroitin 6-sulfate, causing skeletal dysplasia and cardiopulmonary complications. Current enzyme replacement therapy does not impact the bone manifestation of the disease, supporting that new therapeutic alternatives are required. We previously demonstrated the suitability of the CRISPR-nCas9 system to rescue the phenotype of human MPS IVA fibroblasts using iron oxide nanoparticles (IONPs) as non-viral vectors. Here, we have extended this strategy to an MPS IVA mouse model by inserting the human GALNS cDNA into the ROSA26 locus. The results showed increased GALNS activity, mono-KS reduction, partial recovery of the bone pathology, and non-IONPs-related toxicity or antibody-mediated immune response activation. This study provides, for the first time, in vivo evidence of the potential of a CRISPR-nCas9-based gene therapy strategy for treating MPS IVA using non-viral vectors as carriers.

Bone Growth Induction in Mucopolysaccharidosis IVA Mouse.

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is caused by a deficiency of the N-acetylgalactosamine-6-sulfate-sulfatase (GALNS) enzyme, leading to the accumulation of glycosaminoglycans (GAG), keratan sulfate (KS) and chondroitin-6-sulfate (C6S), mainly in cartilage and bone. This lysosomal storage disorder (LSD) is characterized by severe systemic skeletal dysplasia. To this date, none of the treatment options for the MPS IVA patients correct bone pathology. Enzyme replacement therapy with elosulfase alpha provides a limited impact on bone growth and skeletal lesions in MPS IVA patients. To improve bone pathology, we propose a novel gene therapy with a small peptide as a growth-promoting agent for MPS IVA. A small molecule in this peptide family has been found to exert biological actions over the cardiovascular system. This work shows that an AAV vector expressing a C-type natriuretic (CNP) peptide induces bone growth in the MPS IVA mouse model. Histopathological analysis showed the induction of chondrocyte proliferation. CNP peptide also changed the pattern of GAG levels in bone and liver. These results suggest the potential for CNP peptide to be used as a treatment in MPS IVA patients.

Human papillomavirus testing using existing nucleic acid testing platforms to screen women for cervical cancer: implementation studies from five sub-Saharan African countries.

OBJECTIVES: To demonstrate acceptability and operational feasibility of introducing human papillomavirus (HPV) testing as a principal cervical cancer screening method in public health programmes in sub-Saharan Africa. SETTING: 45 primary and secondary health clinics in Malawi, Nigeria, Senegal, Uganda and Zimbabwe. PARTICIPANTS: 15 766 women aged 25-54 years presenting at outpatient departments (Senegal only, general population) or at antiretroviral therapy clinics (all other countries, HIV-positive women only). Eligibility criteria followed national guidelines for cervical cancer screening. INTERVENTIONS: HPV testing was offered to eligible women as a primary screening for cervical cancer, and HPV-positive women were referred for visual inspection with acetic acid (VIA), and if lesions identified, received treatment or referral. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcomes were the proportion of HPV-positive women who received results and linked to VIA and the proportion of HPV-positive and VIA-positive women who received treatment. RESULTS: A total of 15 766 women were screened and tested for HPV, among whom 14 564 (92%) had valid results and 4710/14 564 (32%) were HPV positive. 13 837 (95%) of valid results were returned to the clinic and 3376 (72%) of HPV-positive women received results. Of women receiving VIA (n=2735), 715 (26%) were VIA-positive and 622 (87%) received treatment, 75% on the same day as VIA. CONCLUSIONS: HPV testing was found to be feasible across the five study countries in a public health setting, although attrition was seen at several key points in the cascade of care, namely results return to women and linkage to VIA. Once women received VIA, if eligible, the availability of on-site cryotherapy and thermal ablation allowed for same-day treatment. With sufficient resources and supportive infrastructure to ensure linkage to treatment, use of HPV testing for cervical cancer screening as recommended by WHO is a promising model in low-income and middle-income countries.

Facility-based HIV self-testing strategies may substantially and cost-effectively increase the number of men and youth tested for HIV in Malawi: results from an individual-based mathematical model.

INTRODUCTION: Malawi is rapidly closing the gap in achieving the UNAIDS 95-95-95 targets, with 90% of people living with HIV in Malawi aware of their status. As we approach epidemic control, interventions to improve coverage will become more costly. There is, therefore, an urgent need to identify innovative and low-cost strategies to maintain and increase testing coverage without diverting resources from other HIV services. The objective of this study is to model different combinations of facility-based HIV testing modalities and determine the most cost-effective strategy to increase the proportion of men and youth testing for HIV. METHODS: A data-driven individual-based model was parameterized with data from a community-representative survey (socio-demographic, health service utilization and HIV testing history) of men and youth in Malawi (data collected August 2019). In total, 79 different strategies for the implementation of HIV self-testing (HIVST) and provider-initiated-testing-and-counselling at the outpatient department (OPD) were evaluated. Outcomes included percent of men/youth tested for HIV in a 12-month period, cost-effectiveness and human resource requirements. The testing yield was assumed to be constant across the scenarios. RESULTS: Facility-based HIVST offered year-round resulted in the greatest increase in the proportion of men and youth tested in the OPD (from 45% to 72%-83%), was considered cost-saving for HIVST kit priced at $1.00, and generally reduced required personnel as compared to the status quo. At higher HIVST kit prices, and more relaxed eligibility criteria, all scenarios that considered year-round HIVST in the OPD remained on the cost-effectiveness frontier. CONCLUSIONS: Facility-based HIVST is a cost-effective strategy to increase the proportion of men/youth tested for HIV in Malawi and decreases the human resource requirements for HIV testing in the OPD-providing additional healthcare worker time for other priority healthcare activities.

Sex Difference Leads to Differential Gene Expression Patterns and Therapeutic Efficacy in Mucopolysaccharidosis IVA Murine Model Receiving AAV8 Gene Therapy.

Adeno-associated virus (AAV) vector-based therapies can effectively correct some disease pathology in murine models with mucopolysaccharidoses. However, immunogenicity can limit therapeutic effect as immune responses target capsid proteins, transduced cells, and gene therapy products, ultimately resulting in loss of enzyme activity. Inherent differences in male versus female immune response can significantly impact AAV gene transfer. We aim to investigate sex differences in the immune response to AAV gene therapies in mice with mucopolysaccharidosis IVA (MPS IVA). MPS IVA mice, treated with different AAV vectors expressing human N-acetylgalactosamine 6-sulfate sulfatase (GALNS), demonstrated a more robust antibody response in female mice resulting in subsequent decreased GALNS enzyme activity and less therapeutic efficacy in tissue pathology relative to male mice. Under thyroxine-binding globulin promoter, neutralizing antibody titers in female mice were approximately 4.6-fold higher than in male mice, with GALNS enzyme activity levels approximately 6.8-fold lower. Overall, male mice treated with AAV-based gene therapy showed pathological improvement in the femur and tibial growth plates, ligaments, and articular cartilage as determined by contrasting differences in pathology scores compared to females. Cardiac histology revealed a failure to normalize vacuolation in females, in contrast, to complete correction in male mice. These findings promote the need for further determination of sex-based differences in response to AAV-mediated gene therapy related to developing treatments for MPS IVA.

Improved engraftment and therapeutic efficacy by human genome-edited hematopoietic stem cells with Busulfan-based myeloablation.

Autologous hematopoietic stem cell transplantation using genome-edited cells can become a definitive therapy for hematological and non-hematological disorders with neurological involvement. Proof-of-concept studies using human genome-edited hematopoietic stem cells have been hindered by the low efficiency of engraftment of the edited cells in the bone marrow and their modest efficacy in the CNS. To address these challenges, we tested a myeloablative conditioning regimen based on Busulfan in an immunocompromised model of mucopolysaccharidosis type 1. Compared with sub-lethal irradiation, Busulfan conditioning enhanced the engraftment of edited CD34+ cells in the bone marrow, as well the long-term homing and survival of bone-marrow-derived cells in viscera, and in the CNS, resulting in higher transgene expression and biochemical correction in these organs. Edited cell selection using a clinically compatible marker resulted in a population with low engraftment potential. We conclude that conditioning can impact the engraftment of edited hematopoietic stem cells. Furthermore, Busulfan-conditioned recipients have a higher expression of therapeutic proteins in target organs, particularly in the CNS, constituting a better conditioning approach for non-hematological diseases with neurological involvement.

Comparative analysis between self-collected and clinician-collected samples for HPV testing in public health facilities in Zimbabwe.

BACKGROUND: Cervical cancer screening programs that use visual inspection with acetic acid to identify women with pre-cancerous lesions in Zimbabwe have had limited success due to challenges with human resource constraints and patient acceptability. Nucleic acid amplification tests for human papillomavirus (HPV) have been endorsed by the World Health Organization for cervical cancer screening, along with self-collection of samples. As evidence shows self-collected sampling to be acceptable and preferable, Zimbabwe's Ministry of Health and Child Care (MOHCC) required a comparative analysis on the agreement between self- and clinician-collected samples for testing with Hologic Aptima HPV mRNA assay, to determine if self-collected samples could be used as another method to increase coverage of cervical cancer screening programs. METHODS: In four public health facilities in Zimbabwe from July to August 2020, self- and clinician-collected HPV samples were obtained from HIV-positive women aged 30-49 years for HPV testing. RESULTS: A total of 280 self- and clinician-collected samples were tested and results were found to have good agreement (κ: 0.75, 95% CI: 0.66-0.82). HPV prevalence was 43.0% (95% CI: 37.0%-49.3%) for self-samples and 48.0% (95% CI: 41.0%-54.2%) for clinician-samples. CONCLUSIONS: Self-collected sampling had good agreement with clinician-collected and its inclusion in the national cervical cancer screening policy by Zimbabwe's MOHCC is expected to increase testing coverage, especially among underserved communities such as women living with HIV, as well as decentralize access to cervical cancer screening services for lower-level facilities and increase the geographical scope of where HPV testing can be offered through the country.

Activity of daily living in mucopolysaccharidosis IVA patients: Evaluation of therapeutic efficacy.

BACKGROUND: Mucopolysaccharidosis IVA (MPS IVA, also called Morquio A syndrome) is caused by a deficiency of N-acetylglucosamine-6-sulfate sulfatase (GALNS) and results in skeletal dysplasia symptoms such as short stature and abnormal gait. Treatments include enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT), but the effects are limited depending on the age of initiation and clinical phenotype. Thus, this study aims to assess the effects of treatments on MPS IVA patients compared to untreated MPS IVA patients and an age-matched control group. METHODS: We used activity of daily living (ADL) survey with 4 sections: "movement," "movement with cognition," "cognition," and "other MPS symptoms." Lower scores indicate more assistance required. This study included 161 patients, 270 total surveys, and 70 patients with longitudinal data. RESULTS: We describe 134 severe patients and 25 attenuated patients. ERT and HSCT treatment improved only the "other MPS symptoms" section in severe patients. There were no differences between ERT and HSCT severe patient scores. A 19-year-old male patient, who had robust physical training, provided a significant increase in "movement" without treatment, suggesting the importance of exercise. CONCLUSION: Overall, this ADL questionnaire has demonstrated validation and reliability in assessing the MPS IVA patients and therapeutic efficacy.

Frequency of visits to health facilities and HIV services offered to men, Malawi.

OBJECTIVE: To determine how often men in Malawi attend health facilities and if testing for human immunodeficiency virus (HIV) is offered during facility visits. METHODS: We conducted a cross-sectional, community-representative survey of men (15-64 years) from 36 villages in Malawi. We excluded men who ever tested HIV-positive. Primary outcomes were: health facility visits in the past 12 months (for their own health (client visit) or to support the health services of others (guardian visit)); being offered HIV testing during facility visits; and being tested that same day. We disaggregated all results by HIV testing history: tested ≤ 12 months ago, or in need of testing (never tested or tested > 12 months before). FINDINGS: We included 1116 men in the analysis. Mean age was 34 years (standard deviation: 13.2) and 55% (617/1116) of men needed HIV testing. Regarding facility visits, 82% (920/1116) of all men and 70% (429/617) of men in need of testing made at least one facility visit in the past 12 months. Men made a total of 1973 visits (mean two visits): 39% (765/1973) were as guardians and 84% (1657/1973) were to outpatient departments. Among men needing HIV testing, only 7% (30/429) were offered testing during any visit. The most common reason for not testing was not being offered services (37%; 179/487). CONCLUSION: Men in Malawi attend health facilities regularly, but few of those in need of HIV testing are offered testing services. Health screening services should capitalize on men's routine visits to outpatient departments as clients and guardians.

Epidemiology of Mucopolysaccharidoses Update.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by a lysosomal enzyme deficiency or malfunction, which leads to the accumulation of glycosaminoglycans in tissues and organs. If not treated at an early stage, patients have various health problems, affecting their quality of life and life-span. Two therapeutic options for MPS are widely used in practice: enzyme replacement therapy and hematopoietic stem cell transplantation. However, early diagnosis of MPS is crucial, as treatment may be too late to reverse or ameliorate the disease progress. It has been noted that the prevalence of MPS and each subtype varies based on geographic regions and/or ethnic background. Each type of MPS is caused by a wide range of the mutational spectrum, mainly missense mutations. Some mutations were derived from the common founder effect. In the previous study, Khan et al. 2018 have reported the epidemiology of MPS from 22 countries and 16 regions. In this study, we aimed to update the prevalence of MPS across the world. We have collected and investigated 189 publications related to the prevalence of MPS via PubMed as of December 2020. In total, data from 33 countries and 23 regions were compiled and analyzed. Saudi Arabia provided the highest frequency of overall MPS because of regional or consanguineous marriages (or founder effect), followed by Portugal, Brazil, the Netherlands, and Australia. The newborn screening is an efficient and early diagnosis for MPS. MPS I has been approved for newborn screening in the United States. After the newborn screening of MPS I, the frequency of MPS I increased, compared with the past incidence rates. Overall, we conclude that the current identification methods are not enough to recognize all MPS patients, leading to an inaccurate incidence and status. Differences in ethnic background and/or founder effects impact on the frequency of MPS, which affects the prevalence of MPS. Two-tier newborn screening has accelerated early recognition of MPS I, providing an accurate incidence of patients.

Comparative assessment of impact analysis methods applied to large commercial aircraft crash on reinforced concrete containment.

The precise evaluation of the potential damage caused by large commercial aircraft crash into civil structures, especially nuclear power plants (NPPs), has become essential design consideration. In this study, impact of Boeing 767 against rigid wall and outer containment building (reinforced concrete) of an NPP are simulated in ANSYS/LS-DYNA by using both force time history and missile target interaction methods with impact velocities ranging from 100 m/s to 150 m/s. The results show that impact loads, displacements, stresses for concrete and steel reinforcement, and damaged elements are higher in case of force time history method than missile target interaction method, making the former relatively conservative. It is observed that no perforation or scabbing takes place in case of 100 m/s impact speed, thus preventing any potential leakage. With full mass of Boeing 767 and impact velocity slightly above 100 m/s, the outer containment building can prevent local failure modes. At impact velocity higher than 120 m/s, scabbing and perforations are dominant. This concludes that in design and assessment of NPP structures against aircraft loadings, sufficient thickness or consideration of steel plates are essential to account for local failure modes and overall structural integrity. Furthermore, validation and application of detail 3D finite element and material models to full-scale impact analysis have been carried out to expand the existing database. In rigid wall impact analysis, the impact forces and impulses from FE analysis and Riera's method correspond well, which satisfies the recommendations of relevant standards and further ensure the accuracy of results in full-scale impact analysis. The methodology presented in this paper is extremely effective in simulating structural evaluation of full-scale aircraft impact on important facilities such as NPPs.

Mucolipidoses Overview: Past, Present, and Future.

Mucolipidosis II and III (ML II/III) are caused by a deficiency of uridine-diphosphate N-acetylglucosamine: lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. The process is performed by a sequential two-step process: first, GlcNAc-1-phosphotransferase catalyzes the transfer of GlcNAc-1-phosphate to the selected mannose residues on lysosomal enzymes in the cis-Golgi network. The second step removes GlcNAc from lysosomal enzymes by N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (uncovering enzyme) and exposes the mannose 6-phosphate (M6P) residues in the trans-Golgi network, in which the enzymes are targeted to the lysosomes by M6Preceptors. A deficiency of GlcNAc-1-phosphotransferase causes the hypersecretion of lysosomal enzymes out of cells, resulting in a shortage of multiple lysosomal enzymes within lysosomes. Due to a lack of GlcNAc-1-phosphotransferase, the accumulation of cholesterol, phospholipids, glycosaminoglycans (GAGs), and other undegraded substrates occurs in the lysosomes. Clinically, ML II and ML III exhibit quite similar manifestations to mucopolysaccharidoses (MPSs), including specific skeletal deformities known as dysostosis multiplex and gingival hyperplasia. The life expectancy is less than 10 years in the severe type, and there is no definitive treatment for this disease. In this review, we have described the updated diagnosis and therapy on ML II/III.

Early access to antiretroviral therapy versus standard of care among HIV-positive participants in Eswatini in the public health sector: the MaxART stepped-wedge randomized controlled trial.

INTRODUCTION: The WHO recommends antiretroviral treatment (ART) for all HIV-positive patients regardless of CD4 count or disease stage, referred to as "Early Access to ART for All" (EAAA). The health systems effects of EAAA implementation are unknown. This trial was implemented in a government-managed public health system with the aim to examine the "real world" impact of EAAA on care retention and viral suppression. METHODS: In this stepped-wedge randomized controlled trial, 14 public sector health facilities in Eswatini were paired and randomly assigned to stepwise transition from standard of care (SoC) to EAAA. ART-naïve participants ≥18 years who were not pregnant or breastfeeding were eligible for enrolment. We used Cox proportional hazard models with censoring at clinic transition to estimate the effects of EAAA on retention in care and retention and viral suppression combined. RESULTS: Between September 2014 and August 2017, 3405 participants were enrolled. In SoC and EAAA respectively, 12-month HIV care retention rates were 80% (95% CI: 77 to 83) and 86% (95% CI: 83 to 88). The 12-month combined retention and viral suppression endpoint rates were 44% (95% CI: 40 to 48) under SoC compared to 80% (95% CI: 77 to 83) under EAAA. EAAA increased both retention (HR: 1·60, 95% CI: 1·15 to 2·21, p = 0.005) and retention and viral suppression combined (HR: 4.88, 95% CI: 2.96 to 8.05, p < 0.001). We also identified significant gaps in current health systems ability to provide viral load (VL) monitoring with 80% participants in SoC and 66% in EAAA having a missing VL at last contact. CONCLUSIONS: The observed improvement in retention in care and on the combined retention and viral suppression provides an important co-benefit of EAAA to HIV-positive adults themselves, at least in the short term. Our results from this "real world" health systems trial strongly support EAAA for Eswatini and countries with similar HIV epidemics and health systems. VL monitoring needs to be scaled up for appropriate care management.

Getting to 90-90-90: Experiences from the MaxART Early Access to ART for All (EAAA) Trial in Eswatini.

PURPOSE OF REVIEW: The MaxART Consortium-led by the Eswatini Ministry of Health-implemented multiple interventions between 2012 and 2017 to achieve UNAIDS 90-90-90 targets. We summarize key findings from community outreach strategies in support of the first 90 goal, and from the Early Access to ART for All (EAAA) trial on the implementation of a "Treat All" strategy to achieve the second and third 90 goals within a government-managed public health system. RECENT FINDINGS: The MaxART Consortium demonstrated that "Fast Track," a problem-solving approach, was effective at increasing testing coverage in the community. Compared with baseline data at 3 months prior to the start of the Fast Track, there was a 273% proportional increase in HIV tests conducted among adolescent males, adolescent females, and adult men, and 722% over baseline for adolescent males. The MaxART EAAA trial further showed that implementation of the Treat All policy was associated with significant two-fold shorter time from enrollment into care to ART initiation than under the standard CD4+ cell threshold-based treatment guidelines. Finally, through the MaxART trial, Eswatini was able to identify areas for further investment, including addressing the system-side barriers to routine viral load monitoring, and designing and implementing innovative community-based approaches to reach individuals who were not more routinely accessing HIV testing and counseling services. As low- and middle-income countries adopt the Treat All approach in their national HIV care and treatment guidelines, further implementation science research is needed to understand and address the system-level barriers to achieving the benefits of Treat All for HIV-infected individuals and those at risk.

Liver-Targeted AAV8 Gene Therapy Ameliorates Skeletal and Cardiovascular Pathology in a Mucopolysaccharidosis IVA Murine Model.

Mucopolysaccharidosis type IVA (MPS IVA) is due to the deficiency of GALNS (N-acetylgalactosamine 6-sulfate sulfatase) and is characterized by systemic skeletal dysplasia. We have evaluated adeno-associated virus 8 (AAV8) vectors expressing different forms of human GALNS under a liver-specific promoter. The vectors were delivered intravenously into 4-week-old MPS IVA knockout (KO) and immune tolerant (MTOL) mice at a dose of 5 × 1013 genome copies (GC)/kg. These mice were monitored for 12 weeks post-injection. GALNS enzyme activity was elevated significantly in plasma of all treated mice at 2 weeks post-injection. The activity observed was 4- to 19-fold higher than that in wild-type mice and was maintained throughout the monitoring period. Treatment with AAV vectors resulted in a reduction of keratan sulfate (KS) levels in plasma to normal levels 2 weeks post-injection, which were maintained until necropsy. Both vectors reduced the storage in articular cartilage, ligaments, and meniscus surrounding articular cartilage and growth plate region as well as heart muscle and valves. Our results suggest that the continuous presence of high levels of circulating enzyme increases the penetration into bone and heart and reduces the KS level, thereby improving storage in these regions. The current data support a strategy for developing a novel treatment to address the bone and heart disease in MPS IVA using AAV gene therapy.

Neonatal combination therapy improves some of the clinical manifestations in the Mucopolysaccharidosis type I murine model.

Mucopolysaccharidosis type I (MPS-I), a lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase enzyme, results in the progressive accumulation of glycosaminoglycans and consequent multiorgan dysfunction. Despite the effectiveness of hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) in correcting clinical manifestations related to visceral organs, complete improvement of musculoskeletal and neurocognitive defects remains an unmet challenge and provides an impact on patients' quality of life. We tested the therapeutic efficacy of combining HSCT and ERT in the neonatal period. Using a mouse model of MPS-I, we demonstrated that the combination therapy improved clinical manifestations in organs usually refractory to current treatment. Moreover, combination with HSCT prevented the production of anti-IDUA antibodies that negatively impact ERT efficacy. The added benefits of combining both treatments also resulted in a reduction of skeletal anomalies and a trend towards decreased neuroinflammation and metabolic abnormalities. As currently there are limited therapeutic options for MPS-I patients, our findings suggest that the combination of HSCT and ERT during the neonatal period may provide a further step forward in the treatment of this rare disease.

Brain Pathology in Mucopolysaccharidoses (MPS) Patients with Neurological Forms.

Mucopolysaccharidoses (MPS) are the group of lysosomal storage disorders caused by deficiencies of enzymes involved in the stepwise degradation of glycosaminoglycans. To identify brain pathology common for neurological MPS, we conducted a comprehensive analysis of brain cortex tissues from post-mortem autopsy materials of eight patients affected with MPS I, II, IIIA, IIIC, and IIID, and age-matched controls. Frozen brain tissues were analyzed for the abundance of glycosaminoglycans (heparan, dermatan, and keratan sulfates) by LC-MS/MS, glycosphingolipids by normal phase HPLC, and presence of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor superfamily member 10 (TNFSF10) by Western blotting. Fixed tissues were stained for the markers for microgliosis, astrogliosis, misfolded proteins, impaired autophagy, and GM2ganglioside. Our results demonstrate that increase of heparan sulfate, decrease of keratan sulfate, and storage of simple  monosialogangliosides 2 and 3 (GM2 and GM3) as well as the neutralglycosphingolipid, LacCer, together with neuroinflammation and neuronal accumulation of misfolded proteins are the hallmarks of brain pathology in MPS patients. These biomarkers aresimilar to those reported in the corresponding mouse models, suggesting that the pathological mechanism is common for all neurological MPS in humans and mice.

Integration of radiology in the modular system at the undergraduate level.

BACKGROUND: Integration of Radiology is challenging because in the traditional system it is introduced with a clinical subject while in an integrated curriculum, vertical integration of Radiology is done with anatomy in the first year and with pathology, forensic medicine, ophthalmology, ENT, gynaecology surgery, and medicine till the final year. This study was done with the purpose to evaluate Radiology teaching in an integrated curriculum in undergraduate students of Azad Jammu Kashmir Medical College/Sheikh Khalifa Bin Zaid Hospital Muzaffarabad. METHODS: This study was done to determine student's perceptions regarding Radiology teaching at Azad Jammu Kashmir Medical College (AJKMC), starting from the foundation module of the first year till the final year. It was a descriptive cross-sectional type conducted in the Radiology department of AJKMC. The study duration was six months. Students of final year and recent graduates were included in the study. All the information was collected on pro forma. Pro forma included 11 structured, close-ended, quantitative types of questions. Five points Likert scale was given starting from strongly agree to strongly disagree. Data was analysed by descriptive statistics. RESULTS: In 100 students who gave feedback, the age range was from 23 to 26 year. Male students were 32 (32%) and female 68(68 %). 70% of students agreed and 14% strongly agreed for the integration of Radiology at the undergraduate level. Six percent students disagreed with the integration of radiology at the undergraduate level. CONCLUSION: Integration of Radiology in a modular system for undergraduate students at AJKMC was supported by the majority of participants.