Bypassing the Immortalization Barrier: The Rise of In-silico Gene Expression Engineering using Generative AI

In the fascinating world of biological research, technological advances and innovative methods continue to redefine our understanding of life’s complexities. While the traditional “wet lab” techniques have been instrumental in our knowledge expansion, the in-silico or computational approach to understanding gene expression, especially in oncology and neurodegenerative diseases, is gaining momentum.

This evolution is primarily driven by the inherent challenges and limitations associated with cell immortalization, a crucial process in wet lab research.

What is Cell Immortalization?

Cell immortalization is a process that alters a cell to allow it to divide indefinitely. This unique characteristic makes immortalized cells exceptionally useful for scientific investigations. After all, a constant, renewable supply of cells that are genetically identical allows for reproducible experiments and sustained investigations.

Theoretically, many types of cells can be immortalized for research purposes, but practically it can be quite challenging, and not all cells are equally susceptible to the methods typically used to induce immortalization.

The process of immortalizing a cell involves inducing it to bypass normal cellular controls that limit its lifespan, causing it to continuously divide. The most common techniques for inducing this transformation include viral transformation and the introduction of specific genes that override these controls. For example, introducing the human TERT gene into a cell can lead to the production of telomerase, an enzyme that elongates telomeres (protective caps on the ends of our chromosomes), thus allowing the cell to divide indefinitely.

However, these techniques don’t work on all cell types and can be difficult to implement. Moreover, some cell types, like neurons, are more resistant to standard immortalization techniques because they are terminally differentiated, meaning they have exited the cell cycle and lost their ability to divide.

It’s also important to note that the process of immortalizing a cell can alter its characteristics, sometimes significantly. This is because the mechanisms that limit cell division often play other roles in cell function as well.

For example, p53, a protein often inactivated in immortalized and cancer cells, plays a role in DNA repair, and its loss could lead to an increase in genetic instability. Therefore, while immortalized cells are very useful for certain types of research, they may not always accurately represent the behavior of the cells in a living organism.

The Many Faces of Cell Immortalization

The path to cell immortalization isn’t one-dimensional. It can be achieved in several ways:

Spontaneous Immortalization: A rare occurrence, spontaneous immortalization takes place after many cell culture passages. Overtime, the accumulation of certain mutations can naturally cause cells to bypass the typical cellular division limits.

Viral Immortalization: Certain viruses, like the Epstein-Barr virus, can immortalize cells by carrying genes that disrupt the cell cycle control mechanisms, leading to continuous division.

Genetic Manipulation: Genetic engineering has allowed researchers to manually immortalize cells. The introduction of the TERT gene, for instance, triggers the production of telomerase, an enzyme that extends telomeres, thereby allowing cells to divide endlessly.

The Challenges and Risks of Cell Immortalization

While the concept of an endlessly replicating cell line might seem like a boon for scientific research, the path to immortalization is fraught with challenges and potential alterations to cell characteristics.

First, not all cells are equally susceptible to immortalization. Particularly, certain cell types, like neurons, which are critical to neurodegenerative disease research, are notably resistant to common immortalization techniques due to their terminal differentiation status.

Moreover, in the realm of oncology research, cancer cells themselves are essentially immortalized cells, having bypassed the usual safety checks that curb uncontrolled growth. However, immortalization may not accurately reflect the genetic and molecular landscape of the cancer cells found in a patient’s body. This discrepancy can complicate the interpretation of research findings and their potential clinical applications.

Secondly, the process of immortalization can fundamentally change the cell’s behavior. Bypassing cellular checkpoints often leads to genetic instability, which could limit the predictive validity of the cell line in question. This aspect is especially critical when modeling complex diseases such as cancer and neurodegenerative disorders, where multiple genetic and environmental factors interplay.

A Closer Look at Strategies

In the realm of cell culture, achieving immortalization — the ability of cells to divide indefinitely — is akin to finding the Holy Grail. Several strategies have been developed to immortalize mammalian cells in culture conditions, each with its unique advantages and hurdles.

Harnessing Viral Genes: The Power of SV40 T Antigen: A prevalent method for cell immortalization involves the use of viral genes, such as the Simian Virus 40 (SV40) T antigen. The allure of SV40 T antigen lies in its simplicity and reliability. It has demonstrated its efficiency in immortalizing various cell types, making it a popular choice in scientific endeavors.

The workings of SV40 T antigen in cell immortalization are relatively well understood. This viral gene does more than just induce immortality; it can also stimulate Telomerase activity in the cells it infects, an attribute that further boosts its appeal.

Turning the Tables with Telomerase Reverse Transcriptase (TERT): The most recent addition to the immortalization toolkit is the expression of Telomerase Reverse Transcriptase protein (TERT). This approach is especially beneficial for cells most affected by telomere length, like human cells.

TERT is typically dormant in most somatic cells. However, when human TERT (hTERT) is externally expressed, the cells can maintain ample telomere lengths, successfully dodging replicative senescence. Examination of numerous telomerase-immortalized cell lines confirms that hTERT over-expression leads to immortalization while preserving a stable genotype and essential phenotypic markers.

Yet, hTERT isn’t a one-size-fits-all solution. Over-expression of hTERT fails to immortalize some cell types, notably epithelial cells, and may even cause cell death. But recent breakthroughs have shown that co-expressing the hTERT catalytic subunit with either p53 or RB siRNA can immortalize human primary ovarian epithelial cells, offering a more authentic cell model with a well-defined genetic background.

Over-expression of other genes, such as Ras or Myc T58A mutants, has also been found to immortalize specific primary cell types. Notably, viral genes often achieve immortalization by deactivating tumor suppressor genes like p53 and Rb. These genes can induce a replicative senescent state in cells, so their inactivation is key to achieving immortality.

The Compelling Case for In-silico Gene Expression

Given these challenges, in-silico gene expression emerges as a promising alternative. This computational approach leverages the power of Artificial Intelligence to understand how genes are expressed in different cells and under different conditions.

One of the most significant advantages of in-silico gene expression is the ability to circumvent the issues associated with cell immortalization. Generative AI can accurately predict the gene expression in a variety of cell types, including those resistant to immortalization, providing insights that would be impossible to glean from wet lab experiments alone.

In-silico approaches can model the complex genetic interactions and changes that occur in diseases like cancer and neurodegenerative disorders. Researchers can induce virtual “mutations” and observe the subsequent changes in gene expression, opening a window into the molecular mechanisms underlying these diseases.

Also, the proliferation of high-throughput sequencing technologies has led to an explosion of genomic data. In-silico methods are uniquely poised to make sense of this data deluge, identifying patterns and associations that would be impossible to detect manually.

At Cognit, we are focused on in-silico gene expression engineering to enable a perturbative orcale that not only predicts gene expression given mutations in enhacers and promoters in distal regulatory elements, but we can also allow CRISPR-CAS like experiments in-silico.

An Immortal Future?

Despite the significant strides in cell immortalization techniques, the challenges associated with this process underscore the importance of leveraging computational methods in understanding gene expression. In-silico approaches, with their capacity to process vast amounts of data, model complex interactions, and simulate different scenarios, represent an indispensable tool in the modern biologist’s arsenal.

Cell immortalization will continue to play a vital role in biological research. Still, the future may lie in a hybrid approach, combining the strengths of wet lab experiments with the power of in-silico methods. By capitalizing on these complementary strategies, we stand a better chance at unraveling the complexities of diseases like cancer and neurodegenerative disorders, bringing us one step closer to effective treatments and cures.