BIOCHEMISTRY, BIOMEDICINE & PHARMACEUTICS

AAV5 mediated delivery of micro-RNA for treatment of ALS and FTD with C9orf72 G4C2 expansion Amyotrophic lateral sclerosis (ALS) is a life-threatening neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons, leading to muscle atrophy and paralysis. A significant number of ALS patients also develop frontal temporal dementia (FTD) caused by progressive degeneration of frontal and temporal lobes. There are no disease-modifying drug and most patients die within 3-5 years after the onset of the disease. The most common genetic cause of ALS and FTD is an expansion of G4C2 repeats (> one hundred repeats) in the first intronic site of the  c9orf72 gene present on chromosome 9.   The two underlying pathogenic mechanisms have been proposed for c9orf72 related neurotoxicity. First, the loss of function of c9orf72 caused by G4C2 expansion disrupts mRNA splicing and decreased protein expression resulting in neurotoxicity. But t...

CRISPR is a gene editing tool that are being explored as a potential gene therapy candidate for gene deletions, corrections etc. The high efficiency and specificity of the CRISPR enable its global use in basic research and drug discovery research. The CRISPR consists of two components a) Cas9, a protein that has a nuclease activity b) Guide RNA that recognizes the target sequence, and enables cleavage of target sequence catalyzed by Cas9. Cas9 is a protein that is originally isolated from bacteria Staphylococcus aureus and Streptococcus pyogenes. Because Cas9 is originated from human pathogenic bacteria, the prior exposure to the protein may result in pre-existing antibodies and T cell response against Cas9. The pre-existing immunogenicity against the Cas9 poses a risk impacting the safety and efficacy of CRISPR mediated gene therapy. Recent studies have reported that the prevalence of pre-existing antibodies against Cas9 proteins. Simhari et al. 2018 reported the prevalence of a...

Vestronidase alfa, an enzyme replacement therapy, is an approved drug for the treatment of MPS VII. The approval of this drug has set a precedent for future drug development for the regulators and drug developers. First, the pivotal clinical trial was conducted to evaluate the totality of the clinical data for determining the efficacy of the treatment, therefore, no primary clinical endpoint was established. Second, the drug obtains approval positive outcome on the secondary endpoint; changes in the urinary glycosaminoglycan, from placebo control. The glycosaminoglycan is also the pharmacodynamic biomarker that reflected the efficacy of the drug. Third, the novel multi-domain clinical responder index was designed to evaluate the treatment outcomes and included six minutes walk test (6MWT), forced vital capacity (FVC), shoulder range motion, visual acuity, BOT-2 fine, and gross motor activity. While MRDI evaluation per subject suggested the stabilization of clinical condition if no...

Immune response associated adverse events and hypersensitivity reactions are the major safety concerns related to enzyme replacement therapies. The immunogenicity against these therapeutic enzymes depends on structural similarities, manufacturing processes, formulation, and impurities etc. The drug developer has developed strategies to mitigate manufacturing related immunogenicity risk with improved processes such as optimization of the protein sequence, mammalian cell lines to express and purify recombinant human enzymes. Despite these efforts, the immunogenicity and anti-drug antibody generation against these therapeutic proteins are heterogeneous among the populations. In the same dose cohort, one subject may produce higher anti-drug antibodies titer with lesser efficacy. In contrast, another subject may have less or no anti-drug antibodies with no impact on drug efficacy. Identify and characterizing these individual factors would enable a better understanding of individual fact...

Adeno associated virus (AAV) is a naturally occurring single-stranded DNA virus that belongs to the parvovirus family. The commonly used viral vectors for gene therapy are derived from naturally occurring AAVs. These vectors are genetically modified/engineered that do not contain and devoid of genes encoding viral proteins critical for their viral replication. The viral gene is replaced with the transgene of interest is packaged into the AAV viral capsid and administration to humans. The AAV viral vectors enable attachment and entry of transgene into the target cells. Once inside the cell nucleus, the transgene remains in the episomal form and expresses the gene of interest. Although recent evidence has shown that long-term stable expression of these transgenes can be achieved in pre-clinical models and humans, there is inherent immunogenicity risk associated with adeno-associated vectors which include: -Pre-existing antibodies and T cell response against viral vectors -Neutralizing an...

What are Hemophilia disorders ? Hemophilia is the group of bleeding disorders that are caused by a deficiency of proteins (factors) of the blood clotting cascade. Hemophilia A is caused by the deficiency of factor VIII protein and Hemophilia B is caused by a downstream protein factor IX. The deficiency of the factors VIII and IX affect the clotting cascade, resulting in higher and spontaneous bleeding episodes. The severe bleeding disorder leads to spontaneous bleeding in joints and soft tissue resulting in arthropathy; and increased risk of intravascular and intracranial hemorrhage. Why the development of new treatment paradigm is necessary? The standard treatment for hemophilia (A and B type) is the intravenous infusion of an exogenous clotting protein more than once a week to prevent bleeding. In addition, the development of inhibitors decreases the efficacy of the exogenous replacement factor and the severe hemophilia patients do not respond to these therapies.  There...

Inherited Biallelic RPE65 Mutation-Associated Retinal Dystrophy Inherited retinal dystrophies are a group of rare blinding conditions that are associated with progressive visual dysfunction. This rare genetic disorder is classified based on the phenotype, and the mutation in any one of more than 220 different genes are causal for this rare blinding condition. Retinitis pigmentosa is the most common subgroup of inherited retinal dystrophy characterized by reduced ability to perceive light and progressive loss of visual field. RP is genetically heterogeneous, and mutations in any one of over one hundred genes can cause the phenotype. RPE65 gene encodes all-trans-retinyl ester isomerase, an enzyme crucial to the retinoid cycle Autosomal recessive biallelic mutations in this gene lead to the inability to regenerate 11-cis-retinal, via 11-cis-retinol, in the retinal pigment epithelial cells RPE cells. This impairs the ability of RPE cells to respond to light, which disrupts the vis...