BIOCHEMISTRY, BIOMEDICINE & PHARMACEUTICS

Introduction of Early-Phase Trials For Cell and Gene Therapy All Early-Phase trials are conducted under the IND regulations in 21 CFR Part 312 which emphasize the importance of the assessment of trial risks and the safeguards for trial subjects. For early-phase clinical trials, especially first-in-human trials, the primary objective should be an evaluation of safety (21 CFR 312.21). The FDA has issued guidance on "Consideration for the design of early-phase clinical trials of cellular and Gene Therapy Products."  Click Here For Details

If you are hemophilia patient, scientists, you must have heard about the gene UniQure's ( Nasdaq: QURE ) Etranacogene dezaparvovec gene therapy program for the treatment of Hemophilia B. One subject in the HOPE-B clinical trial was diagnosed with Hepatocellular carcinoma, a form of liver cancer. This incident has generated a flurry of news and pessimism about the future of the gene therapy program and UniQure company. The stock was down 10 to 15% with emotional sell-offs from investors. I will tell you three reasons why UniQure still holds a promise to deliver gene therapy for Hemophilia B patients and everyone needs to take a deep breath and show patience. Three Reason Why Etranacogene dezaparvovec Gene Therapy Still Holds A Promise. Adeno-associated Virus Plaform Uniqure's Hemophilia B Gene Therapy Program utilizes Adeno-Associated Virus Serotype 5 (AAV5) to deliver the gene that encodes a functional copy of Factor IX variant (Padua) into the liver tissues. The genetically...

 UniQure today announced that its hemophilia B gene therapy program, including the pivotal, Phase III HOPE-B study, has been placed on clinical hold by the U.S. Food and Drug Administration (FDA). Patient dosing is complete in each of uniQure’s three hemophilia B gene therapy studies, and there is no plan to enroll or treat additional patients.  Learn More about the HOPE-B clinical trial The clinical hold was initiated following the submission of a safety report in mid-December relating to a possibly related serious adverse event associated with a preliminary diagnosis of hepatocellular carcinoma (HCC), a form of liver cancer, in one patient in the HOPE-B trial that was treated with etranacogene dezaparvovec (AMT-061) in October 2019. The patient has multiple risk factors associated with HCC, including a twenty-five-year history of hepatitis C (HCV), hepatitis B virus (HBV), evidence of non-alcoholic fatty liver disease and advanced age. Chronic infections with hepatitis B and...

   UniQure Highlights  UniQure is delivering on the promise of gene therapy - single treatments with potentially curative results.  UniQure is leveraging AAV based gene delivery platform to rapidly advance a pipeline of proprietary and partnered gene therapies to treat patients with severe genetic diseases.  The AAV5 variant or serotype is utilized to deliver product candidates.  Stock Symbol: QURE Platform:  AAV5 based vector to deliver target genes constructs that encode for specific protein or miRNA.   Q3 2020 Company Highlight Enrolled First Four Patients in Phase I/II Clinical Trial of AMT-130 in Huntington’s Disease Top-Line Data from HOPE-B Pivotal Trial in Hemophilia B Expected Before Year-End Initiated IND-enabling Studies for AMT-150 in Spinocerebellar Ataxia Type 3 (SCA3) Cash equivalent:  Net Loss: $53.8 million, or $1.21 loss per share Therapeutic Area  Hemophilia B Fabry disease Huntington's disease SCA Type 3  An...

    Homology Medicine Highlights Homology Medicine is a genetic medicine company  Utilizes proprietary gene editing and gene therapy technologies into novel treatments for patients with rare diseases.  Based on Positive Data, Advancing pheNIX Gene Therapy Clinical Trial for PKU to Dose Expansion Phase $60 Million Equity Investment from Pfizer Inc. - Unveiled In Vivo Gene Therapy Program for Hunter Syndrome Homology Medicine is a genetic medicine company translating proprietary gene editing and gene therapy technologies into novel treatments for patients with rare diseases. Homology Medicines has a broad, powerful, and proprietary gene editing and gene therapy platform, an experienced and dedicated team, and a commitment to bring new treatments and cures forward for the rare disease community.  Stock Symbol: FIXX Platform The proprietary human hematopoietic stem cell-derived adeno-associated virus vectors (AAVHSCs) to are used to deliver the treatment to patients...

  Highlights of Beam Therapeutics   - Biotechnology company developing genetic medicine  - Use proprietary CRISPR based editing technology - Portfolio includes CAR-T cells for leukemia - Exvivo editing for sickle cell disease - In vivo editing for Wilson Disease - Cash or Cash equivalents  $202.2 million  Beam Therapeutics, launched in 2018, is a biotechnology company developing genetic medicine through the use of CRISPR base editing technology. Beam’s proprietary base editors create precise, predictable, and efficient single base changes, at targeted genomic sequences, without making double-stranded breaks in the DNA. This enables a wide range of potential therapeutic editing strategies that The company is using to advance a diversified portfolio of base editing programs. The company is a values-driven organization committed to its people, cutting-edge science, and a vision of providing life-long cures to patients suffering from serious diseases  Beam Ther...

   European Commission (EC) granted full (standard) market authorization for Libmeldy (autologous CD34+ cells encoding the ARSA gene), a lentiviral vector-based gene therapy approved for the treatment of metachromatic leukodystrophy (MLD), characterized by biallelic mutations in the ARSA gene leading to a reduction of the ARSA enzymatic activity in children with i) late infantile or early juvenile forms, without clinical manifestations of the disease, or  ii) the early juvenile form, with early clinical manifestations of the disease, who still have the ability to walk independently and before the onset of cognitive decline.  MLD is a very rare, fatal genetic disorder caused by mutations in the ARSA gene which lead to neurological damage and developmental regression. In its most severe and common forms, young children rapidly lose the ability to walk, talk, and interact with the world around them, and most pass away before adolescence. Libmeldy becomes the first thera...

  Metachromatic leukodystrophy (MLD) is a rare and life-threatening inherited disease of the body’s metabolic system occurring in approximately one in every 100,000 live births in the U.S. MLD is caused by a mutation in the arylsulfatase-A (ARSA) gene that results in the accumulation of sulfatides in the brain and other areas of the body, including the liver, gallbladder, kidneys, and/or spleen. Over time, the nervous system is damaged, leading to neurological problems such as motor, behavioral and cognitive regression, severe spasticity and seizures. Patients with MLD gradually lose the ability to move, talk, swallow, eat and see. Currently, there are no approved treatments for MLD. In its late infantile form, mortality at 5 years from onset is estimated at 50% and 44% at 10 years for juvenile patients. OTL-200 (autologous CD34+ cell enriched population that contains hematopoietic stem and progenitor cells (HSPC) transduced ex vivo using a lentiviral vector encoding the human aryl...

  Mucopolysaccharidosis type IIIA (MPS-IIIA, also known as Sanfilippo syndrome type A) is a rare and life-threatening metabolic disease. People with MPS-IIIA are born with a mutation in the N-sulphoglucosamine sulphohydrolase (SGSH) gene, which, when healthy, helps the body break down sugar molecules called mucopolysaccharides, including heparan sulfate. The buildup of mucopolysaccharides in the brain and other tissues leads to intellectual disability and loss of motor function. MPS-IIIA occurs in approximately one in every 100,000 live births. Life expectancy of children born with MPS-IIIA is estimated to be between 10-25 years. There are currently no approved treatment options for MPS-IIIA. OTL-201 is an investigational ex vivo autologous hematopoietic stem cell gene therapy being studied for the treatment of MPS-IIIA. It uses a modified virus to insert a functional copy of the SGSH gene into a patient’s cells. Gene Therapy With Modified Autologous Hematopoietic Stem Cells for Pa...

     Highlights - LentiGlobin gene therapy for sickle cell disease (bb1111) is an investigational treatment SCD being developed by BlueBird Bio.  - Designed to add functional copies of a modified form of the β-globin gene (βA-T87Q-globin gene)  - Ongoing Phase 3 clinical study to evaluate the efficacy and safety of the gene therapy candidate - Results from phase 2 study showed durable expression of function copy of β-globin gene (βA-T87Q-globin gene) up to 30 months after infusion - Prevented vasculo-occlusive pain crisis in all subjects treated with Lentiglobin. LentiGlobin for SCD was designed to add functional copies of a modified form of the β-globin gene (βA-T87Q-globin gene) into a patient’s own hematopoietic (blood) stem cells (HSCs). Once patients have the βA-T87Q-globin gene, their red blood cells can produce anti-sickling hemoglobin (HbAT87Q) that decreases the proportion of HbS, with the goal of reducing sickled red blood cells, hemolysis, and other c...

   Highlights  - CTX001 is an investigational ex vivo CRISPR gene-edited therapy that is being evaluated for patients suffering from TDT or severe SCD - CTX001 is being developed under a co-development and co-commercialization agreement between - CRISPR Therapeutics and Vertex. - Received Fast Track Designation from FDA - Clinical studies showed positive results with increased HbF & free of vaso-occlusive crises (Date presented in ASH 2020) CTX001 is an investigational ex vivo CRISPR gene-edited therapy that is being evaluated for patients suffering from TDT or severe SCD in which a patient’s hematopoietic stem cells are engineered to produce high levels of fetal hemoglobin (HbF; hemoglobin F) in red blood cells. HbF is a form of the oxygen-carrying hemoglobin that is naturally present at birth, which then switches to the adult form of hemoglobin. The elevation of HbF by CTX001 has the potential to alleviate transfusion requirements for TDT patients and reduce painful...

  CRISPR/Cas9 is a novel prize-winning gene-editing technology that revolutionized personalized medicine. This technology allows precise, rapid, and long-lasting cure of genetic disorders that otherwise does have no cure.  The recent results from human clinical trials on eye disorders and blood cell disorders have shown that the CRISPR-based technology is safe, and has shown durable effects. With his promising technology three companies are pioneering CRISPR-based treatments.  CRISPR Therapeutics (Nasdaq: CRSP) CRISPR Therapeutics is a leading gene-editing company focused on developing transformative gene-based medicines for serious diseases using its proprietary CRISPR/Cas9 platform. CRISPR/Cas9 is a revolutionary gene-editing technology that allows for precise, directed changes to genomic DNA. CRISPR Therapeutics has established a portfolio of therapeutic programs across a broad range of disease areas including hemoglobinopathies, oncology, regenerative medicine, and ra...

  Preparation of Adeno-Associated Viruses Luciferase Stock (Huttner et al 2003, Orlowski et al 2020) - The day before transfection, triple flasks (total surface area 500 sq.cm) is each seeded with one confluent 15 cm dish of HEK293T/17 (CRL-11268, ATCC) cells and incubated overnight at 37°C and 5% CO2.  - Once 90% confluence is reached, transfection is performed by adding the following to 20 mL of room temperature DMEM: 50 μg of cis-plasmid (firefly luciferase from pCMV-GL3), 150 μg of pDG9 or pDG2 (briefly vortex), and 250 μL of PEI-max (Polysciences) solution (pH 4.5).  - For AAV production, 66 μg of pAV plasmid and 133 μg of pHELPER is used. The solution is vortexed for 10 s and incubated at room temperature for 15 min. The transfection mix is then added to 90 mL of pre-warmed DMEM containing 2% FBS and mixed by swirling. The medium from the triple flasks is removed and replaced with the transfection mixture.  - After 3 days of growth, the cells is harvested by vi...

 ER-Tracker is a cell-permeant, a live-cell stain that is highly selective for the endoplasmic reticulum (ER) that when stained using the protocol provided, the staining pattern is partially retained after fixation with formaldehyde.  ER-Tracker™ Red (BODIPY™ TR Glibenclamide) This stain consists of the red-fluorescent BODIPY® TR dye and glibenclamide. Glibenclamide (glyburide) binds to the sulfonylureas receptor of ATP-sensitive K+ channels which are prominent on ER; the pharmacological activity of glibenclamide could potentially affect ER function. Variable expression of sulfonylureas receptor in some specialized cell types may result in non-ER labeling. Excitation: 587nm Emmision 615 nm ER-Tracker™ Green (BODIPY™ FL Glibenclamide) This stain consists of the green-fluorescent BODIPY® FL dye and glibenclamide. Glibenclamide (glyburide) binds to the sulphonylurea receptors of ATP-sensitive K+ channels which are prominent on ER; the pharmacological activity of glibenclamide cou...

 The LysoTracker® probes consist of a fluorophore linked to a weak base that is only partially protonated at neutral pH. This allows LysoTracker® probes to freely permeate cell membranes enabling them to label live cells. LysoTracker® probes are highly selective for acidic organelles, and unlike dinitrophenyl (DNP), LysoTracker® probes do not require use of a second antibody for detection. LysoTracker® probes are available in a variety of fluorescent colors. LysoTracker® Red DND-99 LysoTracker® Red DND-99 is a red-fluorescent dye for labeling and tracking acidic organelles in live cells. Features of all LysoTracker® probes include: • High selectivity for acidic organelles • Good retention after aldehyde fixation • Effective labeling of live cells at nanomolar concentrations • Live cell imaging • Excitation/Emission: 577/590 nm LysoTracker® Green DND-26   LysoTracker® Green DND-26 is a green fluorescent dye that stains acidic compartments in live cells with excitation/emission ...

  LAMP1 lysosomal associated membrane protein 1 is widely used as Lysosome Marker Lysosomal-associated membrane protein 1 is a glycoprotein from a family of Lysosome-associated membrane glycoproteins. The LAMP-1 glycoprotein is a type I transmembrane protein that resides primarily across lysosomal membranes, and functions to provide selectins with carbohydrate ligands. LAMP proteins are responsible in part for maintaining lysosomal integrity, pH, and catabolism. The expected molecular weight of the protein is approximately 110kDa. Antibodies for detecting LAMP 1 Proteins using Western Blot & Immunofluorescence Analysis (Peer-Reviewed Publications) LAMP1 is routinely used as a lysosome marker and LAMP1-positive organelles are often referred to as lysosomal compartments. Below are the antibodies against LAMP 1 used in the peered reviewed publications. LAMP1 Staining (Rostami et al. 2020) Mouse anti-human LAMP-1 (D401S; Cell Signaling Technology):  Monoclonal antibody is prod...

    Protocol for Western Blot for Detection of Protein of Interest 1. Cell Lysis.  The total protein is extracted from cells and tissues using ice-cold cell lysis reagents such as T-PER Tissue Protein Extraction Reagent (Thermo Scientific, USA) supplemented with 1% Triton X-100, and 1X Mammalian ProteaseArrest inhibitor cocktail (G-Biosciences, St. Louis, MO, USA).  2. Protein Quantification. Protein quantification is performed using the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA) or Pierce protein estimation kit (Thermo Scientific, USA) 3. SDS Page Electrophoressis. Protein lysates (typically 10–20μg) are treated with sample loading buffer containing SDS and reducing agents and incubated at 95-degree celsius for 10 minutes.  Run 4–12% gradient NuPAGE gels (Invitrogen, Carlsbad, CA, USA) for approximately 60 minutes at 130 V.  4. Protein Transfer. Protein transfer is performed using the iBLOT 2 dry transfer apparatus (Life Technologies C...

- Sickle cell disease is an inherited blood disorder of hemoglobin synthesis  - Affects 100,000 individuals in the United States - Financial forecast show approximately 1 billion dollar market with CGAR of 14.3% - Potential curative gene therapy are being Bluebird Bio, Aruvant Therapeutics, CRISPR Therapeutics, Editas Therapeutics, Intellia Therapeutics, Sangamo Biosciences, Homology Medicine - Bluebird Bio has a competitive advantage  Learn More

Qiagen DNeasy Blood & Tissue Kits DNeasy Blood & Tissue Kits provide fast and easy silica-based DNA extraction without phenol or chloroform in convenient spin-column and 96-well-plate formats. Most samples can be directly lysed with proteinase K, eliminating the need for mechanical disruption and reducing hands-on time. Optimized protocols for specific sample types provide reproducible extraction of high-quality DNA for life science, genotyping, and veterinary pathogen research applications. For spin-column or 96-well extraction of total DNA from animal blood and tissues and from cells, yeast, bacteria, or viruses Standardized method for a variety of sample types High yields even from specialized samples High-quality DNA Optimized protocols for a range of starting materials Spin-column and 96-well high-throughput formats NEB Monarch Genomic DNA Purification Kit NEB Monarch Genomic DNA Purification Kit is a universal kit for DNA extraction and purification from a...

           Quantification of Adeno-Associated Viral Vectors by Digital Droplet PCR Droplet digital PCR (ddPCR) for AAV Quantitation Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency. These properties allow accurate quantification of both single-stranded and self-complementary AAV genomes. ddPCR method has demonstrated superior sensitivity on the absolute quantification of single-stranded AAV vector genomes by ddPCR over a standard qPCR assay.  ddPCR is suitable when  qPCR is sensitive to the presence of inhibitors in the PCR reaction  qPCR shows erroneous results with the secondary structure of DNA templates  qPCR is dependent on a DNA standard curve for quantification.  Protocol for ddPCR  Preparation of AAV Genomes for ddPCR The preparation of genomic DN...

What are Adverse Events? Adverse event means any untoward medical occurrence associated with the use of a drug in humans, whether or not considered drug-related. An adverse event (also referred to as an adverse experience) can be any unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom, or disease temporally associated with the use of a drug, and does not imply any judgment about causality.  An adverse event can arise with any use of the drug (e.g., off-label use, use in combination with another drug) and with any route of administration, formulation, or dose, including an overdose.  What is a suspected adverse reaction?  An adverse reaction means any adverse event caused by a drug. Adverse reactions are a subset of all suspected adverse reactions where there is reason to conclude that the drug caused the event.  What is a suspected adverse reaction? Suspected adverse reaction means any adverse event for which there is a reasonable po...