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Short Term and Long Term Safety Monitoring For Gene Editing Therapies

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Ago2 Immunoprecipitation for RISC-siRNA Quantitation

 Ago2 (Argonaute 2) immunoprecipitation (IP) is a technique used to isolate RNA-induced silencing complexes (RISC) from cell lysates. This method allows for the specific enrichment of active RISC complexes bound to small interfering RNA (siRNA) or microRNA (miRNA) within cells. By isolating these complexes, researchers can then quantify the siRNA associated with Ago2, which is an essential step in determining the efficacy of RISC loading and siRNA activity. Here’s a detailed overview of how Ago2 immunoprecipitation is performed for RISC-siRNA quantitation: Steps in Ago2 Immunoprecipitation for RISC-siRNA Quantitation Cell Lysis and Preparation of Lysate : Sample Preparation : Collect cells that have been treated with siRNA, then wash them with cold phosphate-buffered saline (PBS) to remove extracellular contaminants. Lysis : Lyse the cells in a gentle, RNA-preserving lysis buffer that typically includes detergents (e.g., NP-40 or Triton X-100), protease inhibitors, and RNase inhibi...
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Guideline on development and manufacture of lentiviral vectors (CHMP/BWP/2458/03)

The guideline with the reference number "CHMP/BWP/2458/03" pertains to the "Guideline on Development and Manufacture of Lentiviral Vectors." This guideline was developed by the Committee for Medicinal Products for Human Use (CHMP) and the Biotechnology Working Party (BWP) of the European Medicines Agency (EMA). It provides recommendations and regulatory guidance for the development and manufacture of lentiviral vectors, which are widely used in gene therapy and cell therapy applications. Here's an overview of the key points covered in this guideline: 1. Introduction: The guideline begins with an introduction highlighting the increasing importance of lentiviral vectors in advanced therapies and the need for guidance on their development and manufacture. 2. Scope: It defines the scope of the guideline, which covers the development and manufacture of lentiviral vectors intended for use in gene therapy and cell therapy products for human use. 3. Quality and Characte...
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Stem-Loop PCR for siRNA

 Stem-loop PCR is a method often used for detecting and quantifying small RNAs, such as siRNA or miRNA, which are typically difficult to amplify directly due to their short lengths. The method involves the design of a stem-loop reverse transcription (RT) primer, which enhances specificity and stability of the short RNA during the RT-PCR process, allowing for sensitive detection and quantification of the siRNA. Here’s a detailed guide to how stem-loop PCR can be applied to siRNA detection: Key Steps in Stem-Loop PCR for siRNA Designing the Stem-Loop RT Primer : Structure : The stem-loop RT primer consists of a loop region flanked by complementary sequences on either side (the "stem"), which will fold back on itself to form a hairpin structure. Specific Binding Region : A short sequence complementary to the 3’ end of the siRNA is added at the end of the stem-loop primer to ensure specific binding to the siRNA target. Stabilization : The loop structure helps prevent primer-dimer...
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Overview of Cut Point Calculation in the Presence of Pre-existing Antibodies

The process involves statistical methods that account for variations in baseline ADA levels across the study population. Here’s a structured approach to calculate the cut point when there is a pre-existing antibody response: 1. Collect Baseline ADA Samples Sample Population : Collect samples from a representative population of treatment-naïve subjects (typically 50-100 individuals). These baseline samples should reflect the typical range of pre-existing ADA levels within the target patient population. Matrix Type : Use serum or plasma samples, as appropriate for the assay matrix. Time Points : Ideally, collect multiple samples per subject pre-treatment to get a clear baseline. 2. Run Baseline Samples in ADA Assay Perform the ADA assay on all baseline samples, running each sample in triplicate to account for intra-assay variability. Record the response values (e.g., optical density (OD) in ELISA) for each sample. If using multiple replicates, calculate the mean response for each sample....
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ICH Q5D Derivation and characterisation of cell substrates used for production of biotechnological/biological products (CPMP/ICH/294/95)

The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) provides guidelines to ensure the quality, safety, and efficacy of pharmaceutical products. ICH Q5D, as outlined in document CPMP/ICH/294/95, addresses the derivation and characterization of cell substrates used for the production of biotechnological and biological products. Below is a detailed elaboration of ICH Q5D: 1. Purpose of ICH Q5D: ICH Q5D provides guidelines for the establishment of cell substrates used in the production of biotechnological and biological products. The primary goal is to ensure the quality, safety, and consistency of cell substrates to minimize potential risks associated with the final product. 2. Cell Substrate Characterization: The guideline emphasizes the importance of thorough characterization of the cell substrate. This includes the origin of the cells, their history, and any relevant genetic information. Detailed documentation of the cell line...
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