July 7, 2020 - SciWriteGlobal

Jul, 2020

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.

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Jul, 2020

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.

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Apr, 2020

COVID-19: What you need to be careful of?

More than 780000 cases of COVID-19 have been reported across the globe. The new coronavirus outbreak has the attention of the entire world as there are no effective medicines or vaccines yet. The virus originated in the Wuhan city of China and spread to numerous countries in almost no time. This does raise multiple questions like what exactly the virus is. Why its transmission rate is so high? And how an individual can avoid catching it? Well, amid this outbreak we will help you know about the virus in detail. Read ahead to gain a brief insight into the challenges the whole world is suffering from and how we all can overcome it.

Understanding the Coronavirus

Coronaviruses are a group of viruses whose exterior layers reflect a crown-like structure. Corona is a Latin word which means crown. There are various types of viruses in this group but most of them cause very slight illness and cold-like symptoms. The pathogenicity of novel Coronavirus has led researchers and scientists across the world to study more about it as there is very little information in the scientific community. Wuhan city of China is considered to be the epicenter of the disease initially from where it spread to numerous countries in no time. The origin of the virus is still not known but some studies suggest that it has been transmitted to humans from the animals.

Symptoms of COVID-19

The symptoms of the COVID-19 are often confusing as they are somewhat similar to the symptoms of common flu. However, the characteristics difference between them is the presence of high fever, sore throat, dry cough, difficulty in breathing and pneumonia in severe conditions. The incubation time for the development of symptoms is considered to be 14 days due to which people suspected of COVID-19 are suggested to quarantine themselves for this period.

Since very little information is available about the virus, researchers and doctors across the globe are contributing crucial information from the recent cases. It is observed that the virus mainly infects people with low immunity. This is the reason that countries with coronavirus outbreak have high infection and death rate among the elderly population. However, it doesn’t stipulate that young people and children are not prone to it. Having weak immunity irrespective of age can make you prone to infections. Therefore, doctors are prescribing for regular exercise and healthy food as a primary effort to protect yourself from the infection.

The major concern over here with the condition is that at the beginning people might not be able to assess the situation (as the grave) due to the mild symptoms associated. In some cases, people confuse the symptoms with that of normal flu and avoid going to the doctor until it becomes severe. During this period, the patient must have come in contact with say ‘n’ number of people which will further infect other ‘n+y’ number of people. This chain continues as the severe symptoms take 14 days to be physically visible. A point comes where there is a sudden outburst of diseases leading to chaos and panic which is often observed in present cases of countries like China, Italy, Iran, the USA, etc.

Precautions to avoid infections

The transmission of coronavirus from an infected person into the atmosphere takes place through the tiny droplets of a sneeze. These droplets though don’t stay suspended in the air for long, it descends and settles onto the surfaces. It is therefore very crucial for every individual to maintain at least 1m of distance from the infected person. Also, one must avoid touching such infected surfaces and later touching their eyes, nose or face. It is therefore very important to wash your hands thoroughly at regular intervals.
To prevent the transmission of the coronavirus, the doctors also suggest avoiding any kind of physical contact with other people such as avoiding handshakes. In case if you are infected with the disease or even with the normal flu, you must sneeze in your elbows rather than in your palms. You can also use handkerchief or tissue to cover your mouth during the sneeze as these practices reduce the chances of the spread of infectious droplets in the atmosphere.

Way ahead

COVID-19 is a disease that may look normal to you due to its mild symptoms at the initial stage. However, the experience in other countries shows that its completely the opposite and one must take effective care as soon as there are visible symptoms. One of the best ways to prevent the spread is to self isolate yourself and undertake prescribed medications. Researchers and scientists across the globe are working hard to come up with potential vaccines and medicine for the disease. But till then we need to consider the basic precautionary steps to avoid escalation of the situation. So, stay safe and spread awareness among your family to help the world fight this disease together. Let’s not forget we all are together in it and we will come out of it together.

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