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Modification of a Constitutive to Glucose-Responsive Liver-Specific Promoter Resulted in Increased Efficacy of Adeno-Associated Virus Serotype 8-Insulin Gene Therapy of Diabetic Mice
We have previously used a hepatotropic adeno-associated viral (AAV) vector with a modified human insulin gene to treat diabetic mice. The HLP (hybrid liver-specific promoter) used was constitutively active and non-responsive to glucose. In this study, we examined the effects of addition of glucose responsive elements (R3G) and incorporation of a 3′ albumin enhancer (3′iALB) on insulin expression. In comparison with the original promoter, glucose responsiveness was only observed in the modified promoters in vitro with a 36 h lag time before the peak expression. A 50% decrease in the number of viral particles at 5 × 109 vector genome (vg)/mouse was required by AAV8-R3GHLP-hINSco to reduce the blood sugar level to near normoglycemia when compared to the original AAV8-HLP-hINSco that needed 1 × 1010 vg/mouse. The further inclusion of an 860 base-pairs 3′iALB enhancer component in the 3′ untranslated region increased the in vitro gene expression significantly but this increase was not observed when the packaged virus was systemically injected in vivo. The addition of R3G to the HLP promoter in the AAV8-human insulin vector increased the insulin expression and secretion, thereby lowering the required dosage for basal insulin treatment. This in turn reduces the risk of liver toxicity and cost of vector production
Adenovirus-Associated Virus Vector-Mediated Gene Transfer in Hemophilia B
NIHR (RP-PG-0310-1001), the
Medical Research Council, the Katharine Dormandy Trust, the U.K.
Department of Health, NHS Blood and Transplant, the NIHR
Biomedical Research Centers (to University College London Hospital
and University College London), the ASSISI Foundation of
Memphis, the American Lebanese Syrian Associated Charities,
the Howard Hughes Medical Institute, the National Heart, Lung,
and Blood Institute (HL094396), the Royal Free Hospital Charity
Special Trustees Fund 35, the Royal Free Hospital NHS Trust, and
St. Jude Children’s Research Hospita
A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.
Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.This study, including the enrollment of cases, sequencing, and analysis received support from the National Institute for Health Research (NIHR) BioResource–Rare Diseases. The NIHR BioResource is funded by the NIHR (http://www.nihr.ac.uk). Research in the Ouwehand Laboratory is also supported by grants from Bristol-Myers Squibb, the British Heart Foundation, the British Society of Haematology, the European Commission, the MRC, the NIHR, and the Wellcome Trust; the laboratory also receives funding from National Health Service Blood and Transplant (NHSBT). The clinical fellows received funding from the MRC (C.L. and S.K.W.); the NIHR–Rare Diseases Translational Research Collaboration (S. Sivapalaratnam); and the British Society for Haematology and National Health Service Blood and Transplant (T.K.B.).This is the author accepted manuscript. The final version is available from American Society of Hematology via http://dx.doi.org/10.1182/blood-2015-12-688267
Mathematical modelling of gene delivery in patients with haemophilia B
Type B haemophilia is a bleeding disorder resulting from a deficiency of coagulation factor IX (FIX). Although gene therapy is a potentially curative treatment option, optimising the dosing of therapeutic genes for patients remains a challenge. Detailed simulation of gene delivery systems is required for improved understanding of the system. Hence, the purpose of this paper is to develop a modelling framework to predict the physiological response of a subject affected by type B haemophilia to a dose of vector. To address this, an integrated pharmacokinetic/pharmacodynamic (PK/PD) modelling platform was developed based on in vivo clinical data for three patients with severe haemophilia B whose functional plasma levels of FIX are less than 1% of the normal value. The plasma FIX activity was considered as the pharmacological effect while the level of serum alanine aminotransferase (ALT) demonstrated the hepatocellular toxicity. Both an individual-based modelling approach and a population modelling approach were used to estimate the physiological parameters of the developed PK/PD models. The models were then validated using data of the clinical study before being used in a simulation-based modelling approach to provide dosing recommendations. The results obtained from the study demonstrate a good prediction of the pharmacokinetics and pharmacodynamics of the vector. Model-based simulations were subsequently performed to guide initial dose selection in order to provide clinicians with better tools to make the decision-making process simpler for designing more effective treatment plans
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