Jordan Lewis

Jordan is a Chemical Engineering student and undergraduate researcher at the University of Florida. She is passionate about genetic engineering and science communication. Her goal is to portray scientific information in a way that is understandable and beneficial to everyone, whether they are immersed in the scientific community or not.

Categories
Biomedical Research Molecular Biology

Novel “Nearly-endless” Protein Pushes the Boundaries of Molecular Biology

Lab work. // National Cancer Institute on Unsplash.com

Background

Within the past century, biotechnology and molecular biology have seen remarkable progress as a result of explorations into anti-phage bacterial defense mechanisms, such as restriction enzymes and CRISPR-Cas systems. The discovery of restriction enzymes nearly sixty years ago opened the door to precise cutting and manipulation of DNA sequences, thus marking the birth of modern genetic engineering. The accessibility of genome editing ramped up decades later with the development of CRISPR-Cas systems as gene editing tools, revealing unparalleled precision and versatility.

Anti-phage bacterial defense systems have led to some of the most intriguing scientific breakthroughs in the world of biotechnology, from synthetic biology to gene therapies. Despite this, hundreds of additional phage defense systems remain unexplored.

Defense-Associated Reverse Transcriptases

Reverse transcriptases (RTs) are enzymes that catalyze the polymerization of DNA from an RNA template. Defense-associated reverse transcriptases (DRTs) are novel reverse transcriptases involved in bacterial anti-phage defense, however the mechanisms by which DRTs confer anti-phage defense are largely unknown. Characterization of these pathways may elucidate potential diverse biotechnological applications.

DRT2

Recently the function of the defense-associated reverse transcriptase 2 (DRT2), has been discovered. The genes encoding for DRT2 consist of only two components: an RT domain and a noncoding RNA (ncRNA). These minimal systems are sufficient to mount an abortive infection (Abi) response in order to defend against foreign invaders.

Within the defense pathway undertaken by DRT2 a portion of the ncRNA is converted into ssDNA by the RT through rolling-circle reverse transcription (RCRT), resulting in the formation of long cDNA products with concatenated repeats. This cDNA product contains an open-reading-frame (ORF) that lacks any in-frame stop codons and is thus referred to as neo (nearly endless ORF). When under phage infection, the single-stranded cDNA undergoes second-strand synthesis to become dsDNA, which is then transcribed into concatenated mRNA. Following this, the mRNA is translated into a Neo protein which leads to programmed cell dormancy, the mechanism by which the cell defends against bacteriophages.

DRT2 mechanism// Jordan Lewis, heavily inspired by Tang et al.

This defense mechanism highlights novel coding potential in the genome by generating genes from RNA templates, challenging the traditional understanding of how genetic information is stored.

Discussion

While it has been demonstrated that the functional outcome of Neo expression is cell growth arrest, the biological pathway connecting Neo expression to growth arrest remains unclear. Through continuing to pursue the investigation of the Neo protein and its role in the DRT2 defense mechanism, science could gain further insights into newfound complexities in the central dogma of molecular biology may aid in opening the door for future potential medical applications

Resources

Loenen W. et al. (2014) Highlights of the DNA cutters: a short history of the restriction enzymes, Nucleic Acids Res. 42(1):3-19

Jinek M. et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science. 337(6096):816-21.

Georjon H, Bernheim A. (2023) The highly diverse antiphage defence systems of bacteria, Nat Rev Microbiol. 21(10):686-700.

Gonzalez-Delgado A. et al. (2021) Prokaryotic reverse transcriptases: from retroelements to specialized defense systems, FEMS Microbiol Rev. 45(6)

Tang S. et al. (2024) De novo gene synthesis by an antiviral reverse transcriptase, bioRxiv 2024.05.08.593200; doi: https://doi.org/10.1101/2024.05.08.593200

Categories
Biomedical Research

How Bacteriophages Could Stop the Antimicrobial Resistance Crisis

Bacterial colonies on a plate. // CDC on Unsplash.com

Introduction

Antimicrobial resistance (AMR) occurs when bacteria, viruses, fungi, and parasites evolve to become drug-resistant, pushing antibiotics and other antimicrobial medicines to practical obsoletism and thus increasing the difficulty of infection treatment. The prevalence of AMR is a pressing issue, cited by the World Health Organization (WHO) as one of the top 10 global public health threats facing humanity.

A promising threat to AMR is the use of “phage therapy”, a method of utilizing bacteriophages (phages) to eliminate specific bacterial strains.

Phages are viruses of bacteria that evolve to be extremely specialized, targeting only their certain type of bacterial cells and not affecting human cells. This opens a door for phage therapy to be used within the human body against bacterial infections, rather than solely antibiotics.

In the 1940s, the Western world saw chemical-based small-molecule antibiotics usurp potential research into phage therapies. Continued research into the potential of phage therapy allowed members of the former USSR to harness bacteriophages to treat disease. Phage therapy is now commonplace in Eastern Europe, specifically in Russia and Georgia where it is offered over-the-counter.

Dependency upon antibiotics has resulted in a fight against antimicrobial resistance, however, phage therapy may just be the method to bring us out of it.

Advantages of Phage Therapy

Bacteriophages occur abundantly on Earth, from the water of the oceans to the human GI tract. To attack bacterial cells, phages initiate the lytic cycle, wherein they first puncture the bacterium and then inject their own genetic information. The cell becomes a metaphorical warehouse for new phages, producing and assembling phages until the cell is full. Phages within the cell release endolysin, an enzyme that signals the end of the lytic cycle and causes the bacterial cell to explode, releasing brand-new phages.

There are many advantages to employing phage therapy over traditional antibiotic approaches.

Phage viruses bind to a bacterial cell, injecting their genetic material. // Biophoto Associates/SPL

Bactericide:

Phages are bactericidal, meaning they kill their host bacteria. However, some antibiotics are only bacteriostatic and cause a bacterium to become stuck within its stationary phase of growth rather than killing it, leaving behind the viable cell and opening a pathway for potential AMR.

Dosage:

As phages undergo the lytic cycle they self-amplify in an exponential fashion, which could potentially allow for a phage therapy to be effective with single or very low dosages. Lingering in the body after infection is not a worry, as phages only continue to multiply as they are killing bacteria.

Flora Bacteria:

Normal flora bacteria, “good” bacteria of the body, do not generally fall into the extremely specific target ranges of phages, which means they are almost totally unaffected by their presence in the human body. When antibiotics are introduced into the body, however, they tend to wreak havoc on any bacterium they come into contact with, including the normal flora that inhabits our bodies and aids us in many everyday functions.

Resistance:

When bacteria develop resistance to a chemical-based antibiotic it resembles an en masse revolt against one “villain”. Yet, bacteria find it relatively more difficult to develop resistance against phages due to their specific host ranges, since there is no single “villain”. Additionally, as bacteria evolve against phages, the phages have the capability to evolve right along with them, protecting the body against any bacterial strains that develop resistance.

FDA Approval

While phage therapy seems a promising opportunity, the US Food and Drug Administration (FDA) agency has yet to approve any therapies. As phage therapies are classified as biological products by the FDA, any manufacturing needs to adhere to standards like GMP, preclinical research, and all stages of clinical trials. This approval process is a tedious effort, however, as of October 2023 the US has the most phage-related Industry-Sponsored Trials (ISTs) on the worldwide scale, showing there is significant industry-related innovation on this front. Some examples of promising phage therapies in clinical trials within the US include WPP-201 for chronic venous leg ulcers and PreforPro for gastrointestinal distress.

Some of these ISTs are in their third phase of clinical trials, wherein the phage therapies are being tested for large-scale efficiency and comparison to currently market-available competitors. This is the final stage of testing prior to a drug or treatment to be submitted to the FDA for approval.

Despite the many advantages of phage therapy, there is still room for major disadvantages that are avoided through thorough testing prior to FDA approval. Namely, phages possess a narrow host range, inherent biological nature, and unfamiliarity within traditional Western medicine. For these reasons, along with the lengthy approval process, there is still time to go before Americans see phage therapies available to them for treatment.

Although the 1940s saw a shift away from bacteriophage research within Western countries, as time progresses, they could still very well be our savior from the self-inflicted antimicrobial resistance crisis.

References

Carroll-Portillo, A., & Lin, H. C. (2019). Bacteriophage and the innate immune system: Access and signaling. Microorganisms, 7(12), 625. https://doi.org/10.3390/microorganisms7120625

Durr, H. A., & Leipzig, N. D. (2023). Advancements in bacteriophage therapies and delivery for bacterial infection. Materials Advances, 4(5), 1249–1257. https://doi.org/10.1039/d2ma00980c

Furfaro, L. L., Payne, M. S., and Chang, B. J. (2018). Bacteriophage therapy: clinical trials and regulatory hurdles. Front. Cell. Infect. Microbiol. 8:376. doi: 10.3389/fcimb.2018.00376

Ledford, H. (2023, June 26). Why phage viruses could be the key to treating deadly infections – if they can be harnessed safely. Nature News. https://www.nature.com/articles/d41586-023-01982-2

Loc-Carrillo, C., & Abedon, S. T. (2011). Pros and cons of phage therapy. Bacteriophage, 1(2), 111–114. https://doi.org/10.4161/bact.1.2.14590

Strathdee, S. A., Hatfull, G. F., Mutalik, V. K., & Schooley, R. T. (2023). Phage therapy: From biological mechanisms to future directions. Cell, 186(1), 17–31. https://doi.org/10.1016/j.cell.2022.11.017

World Health Organization. (n.d.). Antimicrobial resistance. World Health Organization. https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance

Yang, Q., Le, S., Zhu, T., & Wu, N. (2023, September 6). Regulations of phage therapy across the world. Frontiers. https://www.frontiersin.org/articles/10.3389/fmicb.2023.1250848/full#:~:text=While%20there%20is%20no%20FDA,clinical%20trial%20(Table%201).

Categories
Oncology

How AI Is Revealing New Targets for Cancer Drugs

Woman in cancer treatment. // National Cancer Institute at Unsplash.com

Introduction

Targeted drug therapy has proven to be a highly advantageous approach to cancer treatments, presenting high efficiency and low patient drug resistance. 

Yet, there are drawbacks to the use of targeted drug therapy for cancer, including a lack of identified druggable genomic targets that extend across the patient population. Artificial intelligence algorithms can help researchers better understand carcinogenesis and identify new cancer targets. 

Artificial Intelligence (AI) is a field combining computer science and extensive data sets to perceive, understand, and solve problems.

Two major branches of AI applied biologically are machine learning-based and network-based. Machine learning is an application of AI wherein a computer can learn without direct instruction through mathematical models and pattern recognition. Network-based AI sorts and compares data, providing and compensating different perspectives. 

The Food and Drug Administration (FDA) and the international community have presented an increased interest in trustworthy and ethical AI adoption and innovation.

Applications

The development of multiomics technology is a factor that has bolstered the process of identifying novel anti-cancer targets. Multiomics is an approach to biological analysis that consists of forming data sets out of “omics”, which in the case of cancer are epigenetics, genomics, proteomics, and metabolomics. AI can analyze these data sets to investigate for anti-cancer targets. 

BioRender image by You Et al. Artificial intelligence in cancer target identification and drug discovery. https://doi.org/10.1038/s41392-022-00994-0

Epigenetics

Unlike the changes in the nucleotide sequence characteristic of genetic mutation, epigenetics is similar to an “on-and-off” switch for the expression of specific genetic attributes without altering the genome. Cancer can be caused by both a genetic mutation or an epigenetic signal gone awry. 

A major problem facing epigenetics cancer research is finding specific gene patterns that predict which patients will respond to a cancer treatment. The study of the reversal of epigenetic modifications through AI can give insight into how exactly healthy cells become cancerous and which genetic marker within a patient will respond to cancer-treating drugs.

Genomics

The study of genomics involves the mapping of an entire genome including its structure, function, and evolution, through genome-wide assays such as sequencing. A network-based AI is capable of finding similarities in specific genetic sequences and patterns in their phenotypic expression and interactions. Cancerous biomarkers can be identified through genomic data sets, identifying which genes medical professionals should consider oncogenes of interest.

Proteomics

Proteomics refers to proteins and their interactions within the body. Protein-protein interaction (PPI) research classifies a certain type of protein as “indispensable” and associates them as a major site of disease-causing mutations and drug targeting. In a 2017 study, Ravindran et al. found by analyzing the human PPI data from cancer patients that there are 56 indispensable genes in nine cancers, 46 of which were associated with getting cancer for the first time. This protein interaction data can be harnessed through AI to reveal novel cancer-associated interactions and their potential drug targets. 

Metabolomics

Metabolomics is the study of metabolites: the substrates, intermediates, and products of the human metabolic pathway. A hallmark of cancer cells is that they work to alter these metabolites in order to support their rapid growth, providing energy to their biosynthetic pathways and changing their redox balance. Cancer can be detected through biological AI network analysis due to the presence of certain biomarker metabolites within biofluids, cells, and tissues of the body.

Conclusion

All aforementioned “omics” data can be presented for review in the form of multiomics integration analysis. Varied and interconnected data in a network format allows researchers to study carcinogenesis and drug targeting from an overarching perspective in a multifaceted group of patients. 

While treatment of cancer remains a formidable challenge, rapid technological advances in data collection and analysis through the use of artificial intelligence combined with robust information exchange may prove to be increasingly beneficial to the way cancer drugs are created and tested, leading to potential a safer and healthier world.

References
  • Breakthroughs Staff. (2017, December 12). Treating Cancer by Using Epigenetics, the ‘Software’ of Our Genes | Pfizer. Pfizer News. https://www.pfizer.com/news/articles/treating-cancer-using-epigenetics-%E2%80%98software%E2%80%99-our-genes
  • Guenthoer, J., Lilly, M., Starr, T. N., Dadonaite, B., Lovendahl, K. N., Croft, J. T., Stoddard, C. I., Chohan, V., Ding, S., Ruiz, F., Kopp, M. S., Finzi, A., Bloom, J. D., Chu, H. Y., Lee, K. K., & Overbaugh, J. (2023). Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection. Proceedings of the National Academy of Sciences, 120(23), e2220948120. https://doi.org/10.1073/pnas.2220948120
  • Huang, S., Wang, Z., & Zhao, L. (2021). The Crucial Roles of Intermediate Metabolites in Cancer. Cancer Management and Research, 13, 6291–6307. https://doi.org/10.2147/CMAR.S321433
  • Ravindran, V., V., S., & Bagler, G. (2017). Identification of critical regulatory genes in cancer signaling network using controllability analysis. Physica A: Statistical Mechanics and Its Applications, 474, 134–143. https://doi.org/10.1016/j.physa.2017.01.059
  • U.S. Food and Drug Administration. (2022). Using Artificial Intelligence & Machine Learning in the Development of Drug & Biological Products. https://www.fda.gov/media/167973/download
  • You, Y., Lai, X., Pan, Y., Zheng, H., Vera, J., Liu, S., Deng, S., & Zhang, L. (2022). Artificial intelligence in cancer target identification and drug discovery. Signal Transduction and Targeted Therapy, 7(1), Article 1. https://doi.org/10.1038/s41392-022-00994-0
Categories
Biomedical Research Genetics

First CRISPR-based Gene Therapy Could be Approved in 2023

Photo by Thavis 3D on Unsplash.com

CRISPR-based gene therapies have yet to be approved by the FDA, despite their relative affordability and ease when compared to traditional gene therapies. This may change in 2023, as CRISPR Therapeutics and Vertex Pharmaceuticals announced that their biologics licensing applications (BLAs) to the U.S. Food and Drug Administration (FDA) were completed, including a request for priority review, which would shorten the FDA’s traditional twelve-month review of the application to eight months. This timeline opens the possibility for the first CRISPR gene-edited therapy to be available for interstate commerce within the year.

Background

CRISPR/Cas9 complexes were initially discovered in the natural immune systems of bacteria to protect them from viral invaders. The CRISPR component is a sequence complementary to a specific “target” sequence in a patient’s genome. It is sometimes referred to as the Guide RNA, as it guides the entire complex to the place within the genome where editing will occur. The Cas9 enzyme is the protein commonly depicted as a pair of metaphorical scissors, as it cuts DNA to allow for the insertion/deletion of intended genetic material. In the medical field, CRISPR genetic editing can be harnessed to potentially edit the genomes of individuals affected by currently incurable genetic diseases.

The CRISPR/Cas9 complex binds to and cuts the target sequence. / Javier Zarracina via vox.com

CRISPR/Cas9 is much more accessible than other FDA-approved gene therapies, due to its relative affordability and ease of use. Yet the FDA has shown caution when it comes to its approval. Major limitations of CRISPR-based medicines include:

  • The potential for off-targeting, wherein the complex incorrectly recognizes and binds to a sequence similar to the target sequence.
  • The triggering of the body’s immune response by CRISPR/Cas9, as it originates from bacteria.
  • The multi-faceted ethical concerns that come with genetic editing.

Methods and results

Despite concerns, researchers with CRISPR Therapeutics and Vertex Pharmaceuticals are in the final stages of clinical trials and are up for FDA approval with their CRISPR/Cas9 therapy for genetic blood disorders, called exagamglogene autotemcel (exa-cel).

Patients with sickle cell disease (SCD) and transfusion-dependent beta-thalassemia (TDT) who are participating in these trials have stem cells collected from their own blood. These cells are then edited with CRISPR/Cas9 outside of the body. Once the edited cells are introduced back into the body the patients are treated in accordance with traditional hematopoietic stem cell transplant (HSCT) procedures to establish high levels of fetal hemoglobin (HbF) production. HbF is the protein that carries oxygen throughout the bloodstream during fetal development.

The addition of HbF to a patient with SCD allows for a reduction or potential elimination of vaso-occlusive crises, wherein sickled red blood cells block blood flow to specific tissues, depriving them of oxygen and triggering an extremely painful immune response.

Within patients with TDT, increased levels of HbF reduce or eliminate the life-long dependence on blood transfusions that come with the characteristic severe anemia of the disease.

A doctor drawing blood from a patient. / Nguyen Hiep via Unsplash.com

CRISPR Therapeutics and Vertex Pharmaceuticals are in stage III of clinical trials, assessing both adults and children with SCD/TDT. They presented the adult data from 75 patients (31 with SCD and 44 with TDT) at the European Hematology Association Congress in December 2022.

All of the 31 patients with severe SCD that had been experiencing recurrent vaso-occlusive crises saw an elimination of the crises at their follow-up after exa-cel infusion (follow-up ranging from 2.0 to 32.3 months).

Of the 44 blood-transfusion-dependent patients with TDT, 42 were transfusion-free after exa-cel infusion (follow-up ranging from 1.2 to 37.2 months) and two were at the 75% and 85% marks in transfusion-reduction.

These CRISPR-based therapies show solid potential to change the idea of “incurable” blood diseases.

This research supports the biologics licensing applications (BLA) of CRISPR Therapeutics and Vertex Pharmaceuticals. A BLA is a request to the FDA to introduce a biological product, in this case the exa-cel gene therapy, to the interstate market. Within the BLA, there is a request for Priority Review, which would shorten the FDA’s traditional twelve-month review of the application to eight months, potentially allowing for the first CRISPR gene therapy to be FDA-approved within 2023.

Although the exa-cel CRISPR gene therapy is not approved just yet, it is an exciting innovation for CRISPR research and patients affected by “incurable” genetic diseases.

References
  • Frangoul, H., Altshuler, D., Cappellini, M. D., Chen, Y.-S., Domm, J., Eustace, B. K., Foell, J., de la Fuente, J., Grupp, S., Handgretinger, R., Ho, T. W., Kattamis, A., Kernytsky, A., Lekstrom-Himes, J., Li, A. M., Locatelli, F., Mapara, M. Y., de Montalembert, M., Rondelli, D., … Corbacioglu, S. (2021). CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. New England Journal of Medicine, 384(3), 252–260. https://doi.org/10.1056/nejmoa2031054
  • Kingwell, K. (2023, April 3). First CRISPR therapy seeks landmark approval. Nature News. https://www.nature.com/articles/d41573-023-00050-8
  • Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR-cas9 system. Nature Protocols, 8(11), 2281–2308. https://doi.org/10.1038/nprot.2013.143
  • Vertex and CRISPR therapeutics complete submission of rolling biologics license applications (Blas) to the US FDA for exa-Cel for the treatment of sickle cell disease and transfusion-dependent beta thalassemia. Vertex Pharmaceuticals. (2023, April 3). https://investors.vrtx.com/news-releases/news-release-details/vertex-and-crispr-therapeutics-complete-submission-rolling
  • Vertex and CRISPR therapeutics present new data on more patients with longer follow-up treated with exagamglogene autotemcel (exa-cel) at the 2022 European Hematology Association (EHA) Congress. Vertex Pharmaceuticals. (2022, June 11). https://investors.vrtx.com/news-releases/news-release-details/vertex-and-crispr-therapeutics-present-new-data-more-patients