What if we could treat disease, and potentially even cure it, with a single procedure? This is the promise of gene therapy – to correct the underlying genetic cause of disease. While gene therapy has exciting potential, failures in early clinical trials – due to problems with safety and efficacy – highlighted the need to further understand the underlying science of gene therapies. Find out how NanoTemper is helping researchers to develop safer and more effective gene therapies.

Safety and efficacy lead the path forward

Viral vectors — adeno-associated virus (AAV) and lentivirus — and gene editing technologies are key tools in the development of treatments for several diseases like sickle cell anemia, Huntington’s disease, and some types of cancer. To be successful, these therapies must effectively and safely deliver nucleic acids to the target cell. Characterizing the interactions between viral vector or gene-editing proteins and the target cells is one way to tackle the challenge of improving a therapy’s safety and efficacy.

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Viral vectors

AAV and lentivirus are used as viral vectors to deliver a modified gene to the target cell. Researchers are focused on developing better vectors that target only the right cells, avoid triggering the patient immune response, and effectively deliver the therapy with lasting effect. These goals can be achieved by looking at molecular interactions and profiling virus vectors based on their thermal unfolding.



Increase the effectiveness of CD19 CAR-T cell therapy

CD19 CAR-T cell therapy has shown promising results in treating B-cell malignancies using murine CD19 CAR — but can lead to immune recognition in some patients and make the treatment ineffective. This study sought to find out whether humanized CD19 CAR would resolve this problem. MST was used to measure the affinity between CD19 and the murine or humanized CD19 CAR. The humanized CD19 CAR had a 6-fold greater affinity for its human target which improved the outcome of the therapy when used to treat patients — a higher number of therapeutic T cells with increased anti-tumor activity.

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Differentiate AAV serotypes in any buffer

AAV tropism – which determines the target cell or tissue – differs between AAV serotypes. The thermal stability of the AAV capsid is one parameter that can be used to differentiate between serotypes. In this study, nanoDSF was used to measure the thermal stability of different AAV capsids during the development and production process of AAVs. nanoDSF allowed researchers to rapidly identify AAV serotypes in many different formulation and storage buffers.

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See how changes in production method affect your AAV serotypes

Scale-up of recombinant AAVs is key for manufacturers aiming to get their gene therapies to market. While preclinical AAVs are produced in human-derived HEK293 cells, these do not offer ideal throughput for scale up. This study investigated whether the baculovirus-sf9 system made an appropriate substitute to HEK293s, and in particular whether the post-translational modifications (PTMs) of the insect-cell system were sufficient to prevent an adverse reaction in patients. The group used nanoDSF to characterize the differences between different capsid serotypes, as well as different modes of production for the same serotype.

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Payload delivery

In order to design a good gene therapy vector, researchers must consider not only capsid formation and structure, but also how well it delivers the genetic payload to the cell. Gene therapy optimization requires understanding how much genetic material is loaded into the vector, as well as the mechanism of payload release. Avoiding the loss of DNA or RNA during storage and transport is also a critical concern for those working in scale-up and manufacturing of gene therapy products.



Monitor vector stability and prevent DNA loss in AAVs

Manufacturing a successful gene therapy vector relies on the stability of its capsid. This means not only ensuring the capsids are serotyped properly, but also that none of the genetic material is lost over the course of the manufacturing process. This group used nanoDSF to probe vector stability and relate it to the amount of DNA lost in different storage conditions.

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Learn about infectious viral mechanisms, which can improve our understanding of AAVs


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Gene editing technologies

Gene editing technologies can deliver gene therapies via targeted in vivo genome editing – including gene addition, deletion, and correction. In particular, the CRISPR-Cas9 system presents exciting new possibilities for the treatment of genetic disorders. Researchers are now working to make gene editing tools safer by eliminating off-target editing using Cas9 variants or by looking for alternative delivery systems.



Improve gene editing specificity using alternative Cas9 variants

Gene editing with CRISPR-Cas9 has shown great promise in the treatment of diseases such as sickle cell anemia. However, off-target gene editing has been reported for some Cas9 proteins, like SpCas9. This study used MST to show that a different variant of Cas9, FnCas9, has a higher specificity for its intended target and low off-target binding. FnCas9 was then used to successfully correct sickle cell mutations in the patient-derived pluripotent stem cells.

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Design a more specific nucleic acid delivery system

The use of viral vectors still raises concerns about safety due to off-target delivery of nucleic acids. To improve target specificity, the authors designed a chromatin-based nucleic acid delivery system that incorporates antibodies specific to cell surface elements. The key to the success of this system is the efficient capture of the antibody to the chromatin, and MST was used to quantify the interaction between different antibodies and the chromatin. This allowed them to find the best antibody for constructing a highly efficient and specific chromatin-based delivery system for CRISPR-Cas9 gene editing.

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