Exploring CAR-T-cell therapy using CRISPR technology

Exploring CAR-T-cell therapy using CRISPR technology

Immunotherapy is the lesser-known mainstream treatment for cancer. It has recently been gaining popularity ever since the first chimeric antigen receptor T- (CAR-T) cell therapy was approved for Non-Hodgkin lymphoma in 2017. Currently, numerous CAR-T-cell therapies for a variety of cancers are being granted investigational new drug clearance to enter clinical phases.

The clustered short palindromic repeat or also known as CRISPR associated protein 9 (CRISPR/Cas9) technology plays a crucial role in advancing the CAR-T-cell therapy field, owing to its high efficiency, simplicity, and flexibility. It is an exciting new world out there for CAR-T cell therapy researchers, aiming to term cancer a curable disease. 

What is chimeric antigen receptor T- (CAR-T) cell therapy?

CAR-T cell therapy usually involves engineered T cells that act as synthetic receptors. They typically contain a tumor-specific chimeric antigen (CAR) containing an intracellular domain, an antibody derived targeting extracellular domain and transmembrane domain. The transmembrane domain from CD28 is responsible for providing stability to CAR. 

The T cells programmed with CARs can be used to specifically target and kill antigen-expressing cells without the major histocompatibility complex. Data from studies show that CAR-T-cell therapy has helped in the complete remission of patients diagnosed with a variety of solid and hematologic cancers, especially in relapsing cases of acute lymphoblastic leukemia with a remission rate of 80-100%. 

CRISPR technology in developing CAR-T-therapy:

One of the crucial decisions in designing CAR-T cells is choosing the right DNA template for CAR expression. An appropriate DNA template should be obtained easily and rapidly, containing flexible insert sizes, highly efficient target sites, and low cellular toxicity. For a long time, viral vectors were used, but concerns regarding the integration in the wrong location causing unnecessary diseases, gave rise to CRISPR/Cas9 technology. 

A powerful eukaryotic cell genome editing tool, CRISPR/Cas9 technology makes it possible to insert large genes at the required genetic sites in T cells for successful CAR-T engineering without viral vectors. The two essential components of CRISPR technology include a guide RNA (gRNA) customized to recognize the protospacer on target DNA, and a Cas9 protein to create precise double-stranded breaks (DSBs) for gene mutation. 

DSBs have a unique ability to create two distinct mechanisms for repair. One is through the non-homologous joining, which introduces mutations to DSB sites and the other a homology-directed repair (HDR) mechanism which makes sure the donor DNA template is accurately placed for the gene knock-in. The HDR mechanism is popular among researchers due to its precision insert of the CAR expression cassette into the T cells. 

Methods employed to prevent allogeneic CAR-T side effects:

The multigene editing capability of CRISPR/Cas9 technology is employed for the potential safety of any allogeneic CAR-T therapy-associated side effects. For example, to prevent any graft Vs host rejection, the general approach would be to knock out the expression- TCR-αβ of T cells. To function, TCR-αβ requires both α- and β-chains. The α-chains encoding TCR-α can be knocked out using CRISPR gRNA. From previous studies, it can be noticed that when CAR placed under endonuclease transcriptional regulation, it leads to continued T cell function and a delay in cell exhaustion. 

Host Vs graft disease can also be avoided by knocking out β2-microglobulin, an essential part of the major histocompatibility complex class I molecules, using CRISPR to stop the surface antigen presentation. The gRNAs have also been shown to target immune inhibitory receptors enhancing the antitumor activity of CAR-T-cells. 

Future Perspective:

The remarkable use of CAR-T cells in the remission of advanced malignancies is promoting the rapid growth in developing smarter and commercialized CAR-T therapies. The CRISPR/Cas9 genome editing technology promises a hopeful next-generation CAR-T cell product by adding novel CAR-T cells knockout and inducible safe switches to avoid self-killing. 

However, there are certain concerns regarding the technology which includes off-target effects, causing random mutations. Multiple strategies such as optimized gRNA design, careful selection of target sites, prior off-target detection assays should be attempted to minimize the risks. 

By technical progress to avoid mutations, and improved delivery efficiency, CRISPR/Cas9 mediated T cell engineering holds great promise. Currently, experts are working on exploring other CRISPR/Cas 9 gene targets for multiplex editing for potentially developing an optimal off-shelf allogeneic CAR-T cells products as universal treatment options.


Versatile use of Bacterial nanocellulose for wound healing applications

Versatile use of Bacterial nanocellulose for wound healing applications

Over the years, several therapeutic options have been available for wound and burn treatment. The urgent need for better strategies to accelerate treatment leaves more scope for therapeutic improvement in this field. 

Cellulose is one of the most naturally occurring polymers from renewable sources. Occurring in the form of a linear homo-polysaccharide it consists of β‐d‐glucopyranose units linked by β‐1,4 glycosidic bonds. In modern times, bacteria is one of the commonly used sources for producing cellulose also known as bacterial cellulose. Recently, experts have been playing around with the idea of bacterial Nanocellulose (BNC), cellulose constructed using nano-engineering. 

Bacterial nanocellulose matrix has outstanding mechanical and physical properties courtesy of its unique 3D structure. BNC aggregates to form long fibrils, providing room for high elasticity, surface area, and resistance. Such intrinsic characteristics make it the best choice for wound dressings or protecting injured tissues. It does help that BNC is also non-carcinogenic, non-toxic, and biocompatible. 

Bacterial nanocellulose in wound healing:

It is well known that the largest organ of the body is skin. In its native state, the skin is usually dry and acidic in nature. Altered skin integrity is usually caused by systemic factors such as nutrition, among others. When an individual suffers from serious skin damage due to an accident or disease, a complex series of the biological processes are involved in restoring the lost skin. 

A perfect wound dressing must be able to retain moisture and allow oxygen exchange accelerating healing time and preventing infection. Experts consider BNC to be one of the most suitable materials for wound treatments due to its characteristics such as favorable mechanical properties, chemical purity, and water-absorbing capacity. BNC in its natural state has consistently shown great capacity to stimulate wound healing. To further improve the healing effect of BNC, the material can be combined with natural additives such as proteins, glycosides among others, to improve the mechanical strength and cellulose-based dressings with antimicrobial properties. 

Incorporating mesenchymal stem cells into bacterial nanocellulose:

One of the recent strategies to improve BNC wound dressing is the incorporation of mesenchymal stem cells (MSC) in the matrix. MSCs are adult pluripotent stem cells that are expected to integrate into the victim’s tissue and promote regeneration of the damaged tissue.

Several studies prove that MSC can evolve into various cell types including muscle bone and cartilage. They have a great capacity to self-renew while maintaining its integrity, an essential feature needed to improve the wound healing process, and inducing re-epithelization of the wound. 

Genetically engineered bacterial nano cellulose:

Genetic engineering of the BNC is currently being explored with an aim to optimize the properties of the matrix and the cost-effectiveness of the manufacturing process. Previously, strain improvement was performed through the transfer of BNC related gene determinants to a previously prepared “cell factory” organism. This was done to produce a heterologous expression of genes.

Recently a study used a small RNA (sRNA) interference system to improve the native cellulose production path. The constitutive production of the BNC was shut off to prevent any mutants, a common phenomenon in a well-aerated environment. This was replaced by expression vectors to functionalize BNC with specific proteins, by fusing the genes encoding the protein of interest to the short nanocellulose binding domains. 

Challenges and Future Directions:

Using nano engineering in the field of tissue engineering has opened up a lot of new prospects. Experts are looking to develop BNC based out of commercial 3D printing materials, as an alternative to the chemical products such as resins, synthetic thickeners, and plastics, Another added advantage of 3D printed BNC is the possibility of creating flexible and adjustable dressings. The option of 3D printed nanofiber based bacterial cellulose can also offer an opportunity to develop wearable biomedical devices as sensors and drug-releasing materials, to monitor the patient’s wounds constantly. 

Another interesting discovery on the works is the creation of transparent wound coverings using nanocellulose. This discovery will allow optical real-time monitoring of wound healing and help in diagnostics of viral infections and inflammations in chronic wounds. 

In conclusion, although BNC has made substantial progress in the field of tissue engineering, one of the common drawbacks is the non-degradability of nanocellulose in human organisms. This could potentially lead to scar formations and other complications when intended for direct use. However, artificially constructed skin can be used for experimental studies, such as metabolism, and vascularization of skin tissue.

Currently, there are no materials yet to be found that can fully capture the intricates of the native tissue to restore function at an ideal level. The remaining challenge will be to innovate new composite materials using nanoscale engineering to produce fully biomimetic tissues. As the complexity of applications increase, an active remodeling of the existing scaffolds will be required.