What's new

What's New

11 | Oct | 2022

Chinese

CRISPR gene editing has been established as one of the most powerful tools to study disease biology and is also being broadly tested as a therapeutic platform to correct disease causing genetic defects. The CRISPR system was first described in 2012 and in the past decade, the platform has been extensively used for various drug discovery applications including disease biology research, preclinical model development and drug development1. Currently, there are several active clinical trials in various phases that use CRISPR to modify genes in cell therapies or directly edit genes in vivo across seven different disease areas: blood disorders, cancers, ocular diseases, diabetes, infectious diseases, inflammatory diseases, and protein-folding disorders2.

The CRISPR-Cas9 system edits DNA by introducing double stranded breaks to remove or replace genes. However, the creation of double stranded breaks has been shown to trigger the rearrangement of genomic DNA sequences, so using CRISPR as a therapeutic approach carries the risk of inducing significant systemic health issues or cause the development of another disease3. It has long been known that mammalian genomes have several mobile genetic elements called transposons and retrotransposons. These elements are nucleic sequences that have the ability to insert themselves into double stranded DNA breaks and have been associated with disease development including cancers3. Transposons are DNA elements that are inserted into genomic DNA that have double stranded breaks using a cut and paste approach, while retrotransposons are RNA elements that undergo reverse transcription to form DNA elements that are then pasted into genomic DNA. Due to the active transposition of the genetic elements in DNA sequences that have double stranded breaks, there are significant concerns about the short- and long-term safety of using CRISPR based gene therapies.

An alternative gene editing approach called base editing was described in 2016 by David Liu and his colleagues at the Broad Institute4. Base editing is a highly targeted approach that combines CRISPR-Cas9 and the enzyme, cytidine deaminase, to directly change single DNA bases without introducing double stranded DNA breaks. Cytidine deaminase converts cytidine to uridine that results in a C to T base changes. Similarly, a fusion of CRISPR-Cas9 with adenosine deaminase has been developed to change G to A5. There has been a significant amount of validation and testing of the base editors in the 5 years since they were first described. One of the more interesting in vivo studies were reported in 2021 where base editors were delivered in nonhuman primates to efficiently knockdown PCSK9 gene6, suggesting that base editors can be effectively delivered in vivo. Given the promising data from in vivo studies, it is not surprising that base editing technology has been fast tracked into the clinic as a therapy for specific genetic diseases.

In November 2021, Beam Therapeutics, a biotech company based in Cambridge MA, received FDA approval for the first IND filing for base editing technology to treat sickle cell disease7. The therapeutic candidate is an autologous hematopoietic cell therapy that has been modified using base editing to overexpress fetal hemoglobin to counteract the effect of low oxygenation in patients with sickle cell disease7. Another trial was launched in July 2022 where patients with heterozygous familial hypercholesterolemia (HeFH) will be dosed with lipid nanoparticles containing guide RNA and base editor sequences that induces an A to G base change in the PCSK9 gene that is defective in HeFH patients. The base change inactivates the defective PCSK9 gene and reduces LDL levels in patients that results in lower risk for cardiovascular diseases8. While it is too early to predict the success of base editing technology in humans, the chances of success are increased due to the precision and sophistication of targeting individual nucleotides with minimal impacts on local or genome wide sequences.

References:

1https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356701/

2https://innovativegenomics.org/news/crispr-clinical-trials-2022/

3https://answers.childrenshospital.org/crispr-gene-editing/

4https://www.nature.com/articles/nature17946

5https://pubmed.ncbi.nlm.nih.gov/29160308/

6https://www.nature.com/articles/s41586-021-03534-y

7https://investors.beamtx.com/news-releases/news-release-details/beam-therapeutics-provides-business-and-pipeline-updates-and-0

8https://www.the-scientist.com/news-opinion/first-person-dosed-in-novel-gene-editing-clinical-trial-70223

15 | Sep | 2022

Chinese

It is estimated that about 450,000 people live with a form of spinal cord injury (SCI) in the US, and about 17,000 new SCI cases are diagnosed each year primarily due to automobile accidents, falls or acts of violence1. SCIs are graded across 5 tiers that range from recovery of full mobility to complete paralysis. A significant number of spinal cord injuries are typically irreversible as the central nervous system does not have intrinsic repair capabilities and the pharmacoeconomic impact of SCIs is estimated to be about $9.7B1 largely due to the need for lifelong care and possible complications. Current treatments are limited to steroid treatment, surgery (if applicable) and physical therapy but in many cases, the paralysis and loss of sensation are not reversible resulting in a significant reduction in quality of life. Clearly, there is a need for novel therapies that can repair damaged neurons and reverse paralysis.

There has been active research to identify specific factors that can promote neuron repair after SCI. In one study reported in March 2021, researchers at UT Southwestern Medical Center showed that overexpression of the Sox2 protein increased neuron proliferation in mice that had damaged spinal cords. Interestingly, the new neurons formed connections with the existing neurons which suggests that the newly formed neurons could bridge the damage to the spinal cord2. Overexpression of Sox2 also resulted in the reduced formation of scar tissue which is known to impede functional repair of the damaged tissues3. Another study identified a critical subset of genes that are involved in neuron repair in zebrafish – a group at the University of Edinburgh used synthetic RNA Oligo CRISPR guide RNAs to knock down 350 genes and identified 4 key genes that prevented spinal cord repair including TGF-β14. Zebrafish are a good model to screen multiple genes using phenotypic readouts such as fluorescence.

Recently, an exciting study that used novel scaffolds to deliver target proteins has advanced the search for novel repair therapies for spinal cord injuries. Researchers at Northwestern University developed bioactive scaffolds that carry target proteins to the spinal cord injury site. The scaffold consists of supramolecular polymers that are injected as a liquid but then form a gel like mesh mimicking the extracellular matrix or ECM surrounding the spinal cord tissue. The polymer includes 2 modified peptide sequences to activate the transmembrane receptor β1-integrin and basic fibroblast growth factor 2 receptor (FGFR2). Activating β1-integrin reduces the formation of scar tissue while activating basic FGFR2 increasing angiogenesis5. The peptide sequences in the polymers were modified in the non-bioactive domains to increase motion in the nanofibrils. These signal molecules were shown to “dance” or move around in the nanofibril which enhanced interactions with cell surface receptors resulting in increased cellular signaling that leads to vascularization, neuronal regeneration and survival and most importantly, functional recovery5.

Mice who had been paralyzed to mimic spinal cord injury received one injection of the polymer liquid and were able to walk 4 weeks post injection. The modified peptides were shown to persist around the injury site for a significant amount of time to maximize therapeutic efficacy and then degraded after 12 weeks suggesting that this approach has minimal local or systemic toxicity5. Additionally, supramolecular polymers are connected via weak forces compared to conventional polymers that are connected by strong covalent bonds, so they are more dynamic and can be modified for different disease conditions. They are also biodegradable, which makes them ideal for injection into humans. While most of the studies were done in mice, the supramolecular polymers have been tested in an in vitro human cell culture system where it induced increased downstream signaling suggesting that human cells are responsive.

The Northwestern group hopes to start a clinical trial in 2022 after FDA review and the first data sets from the clinical trial will be the true test on whether the moving peptides in the supramolecular polymers can repair neuronal damage and reverse paralysis and loss of sensation.

References:

1https://www.aans.org/en/Patients/Neurosurgical-Conditions-and-Treatments/Spinal-Cord-Injury

2https://www.fiercebiotech.com/research/healing-spinal-cord-injuries-by-reprogramming-scar-forming-cells

3https://www.nature.com/articles/s41467-019-11707-7

4https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009515

5https://www.science.org/doi/10.1126/science.abh3602

18 | Aug | 2022

Chinese

Cell therapies use functional living cells to manage or cure diseases. Cell therapies can be autologous or allogeneic – autologous cell therapies are personalized where a patient’s cells are isolated, modified and then reintroduced to the patient. Autologous therapies have been clinically successful and 5 therapies that target various cancers have been approved by the FDA1. However, autologous cell therapies have manufacturing challenges including long turnaround times, inconsistent source material and interpatient variability. Additionally, supply logistics can be challenging with autologous therapies. Allogeneic therapies are derived from a single donor source which is used to create a master cell bank. Allogeneic therapies have several advantages since they are off the shelf material that have quick turnaround times and simple shipping logistics. Additionally, there is minimal variability in the source material which allows for more control over the therapy’s performance. However, allogeneic therapies have significant challenges due to transplantation of foreign material2and may induce graft vs host disease (GVHD) or the patient’s immune system may attack and clear the infused cells3. Currently, alternative cell therapy sources to treat specific diseases are being investigated including virus-specific T cells and unconventional T cell types such as CD1 T cells, MR1-restricted T cells, and gamma-delta-TCR T cells3. Virus-specific T cells show high antigen specificity which may be impacted by the introduction of an engineered CAR or chimeric antigen receptor. Additionally, the virus-specific T cells were shown to have reduced efficacy over time. CD1 T cells are also being investigated as potential cell therapy sources as different cell types have been shown to express different CD1 forms. B cells express CD1c but antigen presenting cells have been shown to express all 5 (CD1 a-e) forms. There is active research on the potential of using T cells expressing one or more CD1 forms but this work is currently in early stages. Gamma-delta T cells are a rare type of T cells that been shown to be safe but have limited efficacy. However, gamma-delta T cells seem to be amenable to T cell engineering but more work needs to be done to demonstrate efficacy3.

A new method to generating allogeneic T cells has been gaining interest where induced pluripotent stem cells (iPSCs) are gene edited before differentiation into mature T cells4. In a recent 2021 paper, scientists at Kyoto University engineered iPSCs that had no β2-microglobulin, class-II MHC transactivator (CIITA) or CD155 expression while overexpressing single-chain MHC class-I antigen E5. These T cells were also engineered to express CD20 chimeric receptor and were found to evade immune surveillance in a leukemia/lymphoma mouse model that expressed CD205. Another recent study took a similar approach where β2-microglobulin and CIITA expression were disrupted by CRISPR-Cas9 gene editing and CD47 was overexpressed in mouse iPSCs6. The engineered iPSCs were differentiated into either cardiomyocytes or endothelial cells and were tested in models of peripheral arterial disease, ischemic heart failure and A1AT deficiency related lung disease6. The iPSC derived cardiomyocytes improved heart function while the iPSC-derived engineered endothelial cells improved lung function in the mouse models.

In conclusion, these early studies are proof of concept that iPSCs can be engineered with desirable characteristics prior to differentiation into mature cells that serve as cell therapies. More importantly, the iPSC derived differentiated cells appear to evade immune recognition and have the potential to be developed into hypoimmune allogeneic cell therapies that can treat multiple disease conditions including cancer, heart failure, lung diseases etc.

References:

1https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products

2https://www.sciencedirect.com/topics/engineering/allogeneic

3https://www.frontiersin.org/articles/10.3389/fimmu.2020.583716/full

4https://bioengineeringcommunity.nature.com/posts/induced-pluripotent-stem-cells-as-an-off-the-shelf-supply-of-t-cells

5https://www.nature.com/articles/s41551-021-00730-z

6https://www.pnas.org/content/118/28/e2022091118#ref-4

28 | Jul | 2022

Chinese

Uveitis is also known as inflammation of the eye where the vascularized middle layer of the eye becomes inflamed resulting in tissue damage and in some cases vision loss. Uveitis is estimated to have a prevalence of 1 in 1000 people and most of the cases have inflammation in the anterior or front of the eye1. Anterior uveitis is treatable with corticosteroid eye drops such as prednisolone, which is typically the first line of therapy. However, there are some side effects associated with sustained steroid use including increased incidence of glaucoma or carcinogenesis1. However, patients with posterior uveitis that is deeper in the eye may not benefit from topical steroid eye drops as it is estimated that only about 5% of an eye drop medication penetrates eye tissues2. Systemic steroid therapies are an option but are known to have side effects including metabolic disruptions, disrupted wound healing etc. Therefore, there is a clear need for more targeted uveitis treatments that can penetrate ocular tissues and have limited side effects.

More targeted therapies for uveitis will depend on a deeper understanding of the underlying inflammation mechanisms. The eye is considered to be immune-privileged and this is due, in part, to the expression of IL-27, an anti-inflammatory cytokine in retinal cells1. Environmental triggers, infections or other stimuli can trigger the expression of pro-inflammatory cytokines such as IFN-gamma that trigger a strong inflammatory response and recruitment of specific T-cells. The timing of cytokine expression is also critical to manage the balance of pro- and anti-inflammatory cytokine expression. Given the growing body of information on the immune mechanisms associated with uveitis, several investigators are developing immunomodulatory therapies that can delivered directly to the eye.

Viral and non-viral gene therapies are being developed to treat non-infectious uveitis. Researchers at Eyevensys, a biotech based in France, that is working on ocular therapies delivered via electroporation, developed a non-viral gene therapy plasmid that encoded a fusion protein of the TNF-alpha extracellular domain and the human IgG1 Fc domain3. The plasmid was delivered to the ciliary muscle in the eye via electroporation and was shown to reduce inflammation in two rat models of uveitis – an endotoxin-induced model and an autoimmune model. The plasmid therapy (called pEYS606) is currently in phase I/II clinical trials for safety and dose escalation studies4. A group at North Carolina State University reported the development of an adeno-associated virus (AAV) gene therapy to deliver an immunosuppressive gene (HLA-G or IL-10) via intravitreal injection into the eye5. HLA-G is known to have an immunomodulatory and anti-inflammatory effect and is implicated in protecting the fetus from the maternal immune system. IL-10 is a widely studied anti-inflammatory cytokine that inhibits the function of Th1 cells, natural killer (NK) cells and macrophages. The researchers tested the AAV therapy in a rat model of autoimmune uveitis and showed that there was significant reduction in inflammation over 2 weeks post injection. Importantly, the AAV remained largely in the eye and did not spread systemically resulting in a low neutralizing antibody response5.

These data suggest that gene therapies can be delivered directly to the eye carrying anti-inflammatory payloads and have the potential to be novel and effective treatments for non-infectious uveitis. However, the long-term impact of immunomodulatory gene expression in the eye tissues will need to be evaluated along with any long-term toxicity issues due to viral gene delivery. For now, these therapeutic approaches offer a viable alternative to systemic or topical steroids that are less than ideal to treat non-infectious uveitis.

References:

1https://www.aaojournal.org/article/S0161-6420(13)00777-X/fulltext

2https://pubmed.ncbi.nlm.nih.gov/34452185/

3https://pubmed.ncbi.nlm.nih.gov/30009894/

4https://clinicaltrials.gov/ct2/show/NCT03308045

5https://www.ophthalmologytimes.com/view/aav-gene-therapy-targets-autoimmune-uveitis

05 | Jul | 2022

Chinese

CRISPR gene editing is one of the most powerful tools in disease research with the potential to correct genetic defects that cause diseases. CRISPR-mediated gene correcting can either be used to develop curative therapies or prevent onset of disease by correcting the gene defect very early. CRISPR was first described in 2012 and there has been acceleration in the use of CRISPR gene editing for various applications including developing physiologically relevant preclinical animal models, investigating disease biology, confirming disease drivers and most importantly, correcting genetic mutations to cure disease1. While gene therapy using CRISPR has the potential to correct genetic disease drivers and reverse the course of a disease, it is dependent on accurate and safe targeting to the appropriate tissues or organs. The delivery system also has to have desirable ADME (absorption, distribution, metabolism, excretion) characteristics to ensure that the gene therapy is efficacious with minimal off target or toxic effects.

Gene therapy delivery methods can be broadly classified into two segments – viral and nonviral22. However, some studies on the dark genome matter reported that gene regulatory elements occupy more sequence space than the actual protein coding genes and many single nucleotide polymorphisms (SNPs) are found outside of the protein coding genes2. Viral delivery typically uses AAV (adeno-associated virus), adenovirus or lentivirus to deliver the genes of interest to specific tissues using the intrinsic ability of viruses to infect specific cells and introduce the transgene. Nonviral methods typically use either physical or chemical delivery systems and an example of physical delivery is electroporation where cell permeability is increased using electricity to facilitate transgene entry. Chemical delivery systems range from lipid particles that encapsulate the genetic material to RNA aptamers that bind to the cell surface and facilitate transgene uptake into the cell via endocytosis and other uptake pathways2. Both viral and nonviral delivery systems have benefits and challenges but viral approaches are typically more efficient and have been used for several studies to delivery CRISPR gene editing to specific tissues. AAV is the most favored viral vector as it has been used in several gene therapy trials with acceptable safety profiles. However, there are several issues to be considered when using AAV to deliver CRISPR and one of the major issues is the size limitation for the transgene. AAVs can typically hold less than 5kb of genetic material which is a challenge to deliver the guide RNAs, Cas9 enzyme and donor DNA that are components of CRISPR. One solution is to engineer 2 AAVs that contain the CRISPR components with the requirement that both AAVs will need to infect the same cells for the system to work. An alternative approach is to identify and engineer a smaller Cas9 that works as effectively as regular Cas9 enzymes, and this approach resulted in the identification of smaller potent Cas9 enzymes from staphylococcal bacteria3.

Nevertheless, AAV mediated gene editing delivery has been successfully demonstrated in preclinical models. One of the earlier studies from George Church’s group at Harvard showed that CRISPR-mediated excision of mutated exon 23 in the dystrophin gene allowed the expression of the correct protein in skeletal and cardiac muscle in the mdx mouse model of Duchenne muscular dystrophy4. In another study, James Wilson’s group at the University of Pennsylvania delivered 2 AAVs with CRISPR components into mice to correct a urea cycle disorder that is a rare genetic liver disease5. While studies in preclinical mouse models are encouraging, the ultimate objective is to accurately deliver the CRISPR system in humans. The first in vivo clinical trial that uses viral delivery of CRISPR is being conducted by Editas Medicine to treat Leber congenital amaurosis (LCA), an inherited retinal disease that leads to blindness6. Luxturna, an AAV based gene therapy has been approved for LCA but is not curative and reports of retinal degeneration 1-3 years after treatment have been observed. A curative treatment is being evaluated in the clinic using CRISPR gene editing to remove a specific mutation from the cep290 gene. The therapy is an AAV5 viral vector that contains Cas9 and two guide RNAs6 that is delivered directly into the eye. In the past couple of years, clinical trials for CRISPR mediated gene therapies have been launched including one to cure chronic HIV7. and it will be important to demonstrate the CRISPR can be delivered safely and effectively via systemic delivery. Despite the delivery challenges that still need to be addressed, the combination of improved CRISPR technology and engineered viral vectors will likely be used in several new clinical trials to cure difficult to treat diseases and fix “undruggable” disease drivers.

References:

1https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356701/

2https://www.genengnews.com/insights/viral-or-nonviral-which-is-better-for-crispr-based-therapies/

3https://pubmed.ncbi.nlm.nih.gov/25830891/

4https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924477/

5https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786489/

6https://crisprmedicinenews.com/news/disease-roundup-leber-congenital-amaurosis/

7https://www.clinicaltrialsarena.com/news/crispr-gene-editing-hiv-cure/

09 | Jun | 2022

Chinese

The first human genome was published in 2004 and it took 13 years for the complete genome to be sequenced1. The scientific community was very excited to map all the human genes in the genome and it was estimated at that time, that the human genome encoded 25,000 to 40,000 genes2. However, this number proved to be a significant overestimate, and it is now estimated that our genome has about 19,000-20,000 genes. This accounts for only 2% of the genetic material, and raises an important question – what about the other 98% of our genome that is referred to as the dark genome?

Very little is known about the dark genome but it is thought that gene regulation elements including repeat sequences, enhancers, and non-coding RNAs are encoded in these regions. Research into the dark genome has been limited as the focus of drug discovery and biomedical research has been on identifying and modulating protein encoding genes associated with disease phenotypes3. However, some studies on the dark genome matter reported that gene regulatory elements occupy more sequence space than the actual protein coding genes and many single nucleotide polymorphisms (SNPs) are found outside of the protein coding genes2. These findings suggest that there is a lot of very valuable information and potentially a large number of drug targets that are hidden in the dark genome. Scientists are employing powerful tools like the CRISPR-Cas9 gene editing system to probe the dark genome where they can disrupt specific areas and observe the outcome in human cells. The use of other high throughput tools will also help identify the roles of specific regions of the dark genomes. To date, it is estimated that over 200,000 sequences in the dark genome encode proteins that may be disrupted during disease development4. This is not unexpected as RNA and protein coding sequences have been reported across the genomes of mice neurons, specific fishes and parasites4.

A recent study has shown that genes encoded in the dark genome have a direct impact on disease development. Researchers at the University of Cambridge probed the dark genome to identify new targets for bipolar disorder and schizophrenia, and published their findings at the end of 20214. Both diseases have been shown to have a strong genetic link as the heritability of both diseases conditions is about 70%. However, to date, conventional analyses of known genes have not been able to account for the strong heritability so the researchers probed the dark genome to identify factors associated with these diseases. Since schizophrenia and bipolar disorder have been primarily described in humans and are associated with cognition, the researchers searched for novel open reading frames (nORFs) in specific regions of the genome called human accelerated regions (HARs). HARs appear to be newly evolved genomic regions that are human specific.

The study identified over 3,000 nORFs with about 56 nORFs associated with schizophrenia and 40 nORFs associated with bipolar disorder and the researchers hypothesized that some of these nORF encoded proteins could be viable drug targets. The nORF sequences were also identified in transposable elements (TEs) suggesting that the nORF products played a role in gene regulation. Interestingly, changes in expression level of specific peptides encoded by the nORFs correlated with specific phenotypes including psychosis and suicide. This suggests that specific nORFs could be investigated further to develop disease management therapies. Research and drug target discovery in the dark genome regions is in the early days and it will be important to understand the significance of the nORF encoded proteins in disease development and correlate the nORF expression with the heritability of specific diseases.

If the nORF encoded proteins are shown to be true drug development targets, it is likely that accelerated research into the dark genome may provide answers to several questions on the genetic causes of diseases that have perplexed scientists so far.

References:

1https://www.genome.gov/human-genome-project

2https://www.nature.com/articles/538275a

3https://bioengineeringcommunity.nature.com/posts/d

4https://www.nature.com/articles/s41380-021-01405-6

01 | Jun | 2022

It is estimated that about 20% of US adults live with chronic pain1. and pain management medications range from over-the-counter non-steroidal anti-inflammatory drugs (NSAIDs) for mild pain to opioids for severe or chronic pain. While opioids have been shown to manage pain, they are highly addictive and it is estimated that 8 to 12% of people using opioids to manage pain become addicted2. Additionally, opioid addicts often transition to using other narcotics, resulting in increased substance abuse issues that have a huge impact on the individual and society.

Given the enormous social and pharmacoeconomic challenges associated with pain management, there is a need to develop non-addictive pain therapies especially for severe pain. Several studies have shown that voltage-gated sodium channels play a role in the pain response. Specifically, the Nav1.7 channel is expressed on pain-sensing neurons or nociceptors where they send pain signals in response to membrane depolarization due to tissue damage. The important role of Nav1.7 in the pain response was first shown in 2006 where patients with null mutations had insensitivity to pain3. These patients had nonsense mutations that resulted in truncated Nav1.7 protein expression. Conversely, patients with gain of function mutations in Nav1.7 developed a rare disease called primary erythromelalgia that is characterized by burning pain and redness.

Studies on the mechanism of action of Nav1.7 function in response to gain or loss of function have been linked to channel activation or inactivation states4. Gain of function mutations result in the Nav1.7 channel gating being shifted towards activation resulting in hyperpolarization and chronic pain signals. Conversely, loss of function mutations results in the Nav1.7 channel gating being shifted towards a deactivation state with minimal pain signal. A significant body of research over the past several years has collectively demonstrated the role of Nav1.7 in modulating various pain responses4. Not surprisingly, Nav1.7 has become a target for novel analgesic development. However, developing a novel therapeutic targeting Nav1.7 poses a major challenge – there is significant sequence similarities between various voltage-gated sodium channels so developing a Nav1.7 specific small molecule or monoclonal antibody drug has not been successful.

An alternative approach is gene therapy to selectively inhibit Nav1.7 expression. Recently, a landmark paper was published in Science Translational Medicine that demonstrated the use of CRISPR and zinc finger proteins to silence Nav1.75. The approach taken by the group from the University of California, San Diego is novel in that they use a catalytically inactive Cas9 enzyme that does not cut DNA but binds to the target DNA sequence to inhibit gene expression. This “dead” or inactive Cas9 does not modify the genome and is considered to be safer than the regular CRISPR approach. The dead CRISPR system was tested in multiple mouse models of pain including inflammatory pain and paclitaxel induced neuropathic pain. The inactivation of Nav1.7 showed reduced pain in all animals and this insensitivity was shown to be durable for 15 weeks for the neuropathic pain model and 44 weeks for the inflammatory pain model5. The researchers also repeated the study using zinc finger proteins to block Nav1.7 expression and showed that reduction of Nav1.7 increased insensitivity to pain. The inhibition of Nav1.7 using either the CRISPR or zinc finger proteins showed minimal side effects in mice and did not affect normal behavior or mobility.

The research study authors have named this technology pain LATER (long-lasting analgesia via targeted in vivo epigenetic repression of Nav1.7) and have launched a company called Navega Therapeutics to test this gene therapy in human clinical trials. While these are early days, the targeted inhibition of Nav1.7 using gene therapy is based on robust science and clinical observation and has real potential to become a durable and effective therapy to manage chronic and severe pain. The best news of all – patients may have an option for pain relief without the burden of addiction.

References:

1https://www.cdc.gov/mmwr/volumes/67/wr/mm6736a2.htm

2https://www.drugabuse.gov/drug-topics/opioids/opioid-overdose-crisis

3https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212082/

4https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4950419/

5https://stm.sciencemag.org/content/13/584/eaay9056

28 | Apr | 2022

The RAS family of proteins are GTP/GDP on-off switches that regulate downstream cell signaling. They are some of the most widely studied targets for cancer drug development as about 25% of human cancers reportedly have a RAS mutation. The three RAS genes are KRAS, NRAS and HRAS where KRAS is reported to be the most frequently mutated gene1. Mutations in KRAS result in the switch being permanently turned on resulting in uncontrolled cell proliferation and tumor formation. Mutated KRAS is frequently seen in lung, colorectal and pancreatic caners and the most common mutations are around the glycine residue G122. Despite the detailed knowledge of KRAS mutations, drug developers have not seen much success in developing anti-KRAS therapies targeting G12. Indeed, KRAS has been labeled as an “undruggable” target largely due to the protein structure. KRAS is a smooth ball shaped protein that has one GTP binding pocket, so it was thought that there are no other binding pockets for a small molecule inhibitor to bind.

However, this dogma was challenged in 2013 when a group at the University of California, San Francisco published a paper detailing the crystal structure of KRAS that showed the formation of a new binding pocket beneath the effector binding region called switch-II3. This led to a new strategy for small molecule inhibitors targeting KRAS G12C that bind to active GTP-bound KRAS to change binding preference from GTP to GDP, thus locking KRAS in an off position and shutting down downstream signaling and uncontrolled cell proliferation4. This breakthrough accelerated the hunt for novel small molecules that had the right pharmacological properties and structure. Amgen was the first company to identify a small molecule called AMG 510 or sotorasib in collaboration with Araxes Pharmaceuticals. The company took risks by expediting drug development in both preclinical assay testing and clinical trials as well as starting manufacturing earlier5.

Sotorasib demonstrated good efficacy in a clinical trial of non-small cell lung cancer patients with KRAS G12C mutations along with acceptable side effects5. The drug is formulated as an oral once-daily medication that is ideal for patients. Currently, there are a few clinical trials ongoing combining sotorasib with various other therapies including the PD-1 inhibitor pembrolizumab and docetaxel. Based on the positive outcome data from the non-small cell lung cancer trial, the FDA has approved sotorasib (now known as Lumakras) as the first KRAS targeted therapy for non-small cell lung cancer patients with the G12C mutation6. This patient segment represents 13% of mutations in non-small cell lung cancer, which is a significant number of patients.

Amgen is not the only company working on the KRAS inhibitor. Mirati Therapeutics is also working on a KRAS inhibitor called adagrasib, which locks mutant KRAS in the off conformation and is reported to have higher tumor reduction potential than Lumakras7. Based on the early clinical trial data and growing pharma interest, KRAS inhibitors are shaping up to be breakthrough targeted drug therapies especially in combination with other available therapies to shut down compensatory signaling in tumors. It is important to remember that the development of targeted KRAS inhibitors is a fascinating story that started with a photographic image of an undruggable protein.

References:

1https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869631/

2https://www.mdanderson.org/cancerwise/targeting-the-kras-mutation-for-more-effective-cancer-treatment.h00-159458478.html

3Ostrem, J., Peters, U., Sos, M. et al. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature 503, 548–551 (2013).

4Gentile DR, Rathinaswamy MK, Jenkins ML, Moss SM, Siempelkamp BD, Renslo AR, Burke JE, Shokat KM. Ras Binder Induces a Modified Switch-II Pocket in GTP and GDP States. Cell Chem Biol. 2017 Dec 21;24(12):1455-1466.e14.

5https://www.fiercebiotech.com/biotech/how-a-protein-polaroid-led-amgen-to-cusp-a-cancer-breakthrough-lumakras-8-years

6https://www.fda.gov/news-events/press-announcements/fda-approves-first-targeted-therapy-lung-cancer-mutation-previously-considered-resistant-drug

7https://www.fiercepharma.com/special-report/adagrasib-10-most-anticipated-drug-launches-2021

04 | Apr | 2022

Kidney toxicity or nephrotoxicity is defined as a dysfunction caused by drugs, chemical or environmental agents, and is typically a result of inflammation or obstruction of kidney function. While drug-induced nephrotoxicity can cause acute injuries or chronic diseases, recognizing acute kidney injury is the primary focus when evaluating new drugs. Drug-induced nephrotoxicity ranges from 14 to 26% and about 16% of pediatric patients are hospitalized due to drug-induced kidney injury1. As a part of PK/PD studies, it is critical to understand how drugs circulating in the vasculature are transported into the kidney for excretion. The FDA requires the testing of new drugs to determine drug-drug interactions that includes the principal routes of elimination, the roles that specific transporters play in drug elimination, and the effect of the drug on those transporters2.

The kidneys clear drugs and drug metabolites via a combination of passive glomerular filtration and active tubular secretion. The process of tubular secretion has 2 components: the drug and/or metabolites are taken up from the blood via the basolateral membrane of proximal tubule cells and are then transported into the lumen through the apical membranes. Drug transporters typically fall in two categories: the solute carriers (SLC) that are expressed on the basolateral membrane and apical membranes and ATP binding cassettes (ABC) that are expressed on the apical membrane. Animal models are commonly used for ADME studies but there is increasing interest in in vitro models to study specific endpoints such as kidney toxicity. More complex cell-based models are being developed to study drug-transporter interactions. The models range from simple cell lines that overexpress a single transporter to bioprinted kidney models that recapitulate in vivo kidney function.

Cost-effective cell line-based models have been used to study drug-transporter interactions. The most popular cell lines are Caco-2 (human colorectal cancer) and MDCK (canine kidney) cells. Both cell lines partially recapitulate transporter expression profiles – MDCK cells are able to correctly sort transporters to the apical or basolateral cell membrane, and Caco-2 cells endogenously express several transporter proteins. However, these cell lines do not accurately recapitulate the structure and function of renal proximal tubule cells that are highly polarized columnar cells with specific transporters expressed on the apical and basolateral membranes. Proximal tubule epithelial cells isolated from normal kidney tissues are considered optimal for in vitro assessments of drug-transporter interactions and have been shown the highest accuracy in predicting toxicity of over 40 compounds3. Studies on the kidney chip showed improved expression of uptake and efflux transporters, resulting in accurate and reproducible responses to known transporter inhibitors such as cimetidine4. Currently, several commercially available organ-chip models are available to evaluate drug-transporter interactions in drug toxicity studies. The bioprinted kidney model has complex architecture with proximal tubule epithelial cells lining a lumen, and fluid shear stress is applied to the lumen to mimic glomerular filtration5, and is being evaluated as a scalable model to evaluate kidney toxicity.

Based on the sustained activity in developing more physiologically relevant in vitro toxicity models, the drug development community is moving towards more complex cell models leaving the simple cell line approach in the dust.

References:

1https://bmcnephrol.biomedcentral.com/articles/10.1186/s12882-017-0536-3

2https://www.fda.gov/regulatory-information/search-fda-guidance-documents/vitro-drug-interaction-studies-cytochrome-p450-enzyme-and-transporter-mediated-drug-interactions

3https://pubmed.ncbi.nlm.nih.gov/30076203/

4https://pubmed.ncbi.nlm.nih.gov/23644926/

5https://www.nature.com/articles/srep34845

03 | Mar | 2022

The human brain has a unique ability to adapt and change in response to stimuli, which is called neuronal plasticity.  The modifications are done at the cellular level where neurons are capable of altered transmission through their synapses in response to specific activities or stimuli. Neuronal plasticity is a fundamental property of the central nervous system and is considered to be critical for the brain to function optimally1, as it is involved in cognitive skills, brain development and recovery from trauma or injury1. There are reports that learning a new language changes brain anatomy suggesting that neuronal plasticity is critical for improved learning2. Interestingly, in 2008, meditation was found to change brain structure and function and reduce neural noise, suggesting that meditation like techniques can have positive effects on learning, behavior and neuropsychology3. Neuronal plasticity has become an important area of interest in the context of various diseases including neurodegenerative diseases, traumatic brain injury, stroke and psychological diseases.

Depression is reported to be one of the most widespread psychological conditions and can have a severe impact on a person’s quality of life and wellbeing and in extreme cases, it can result in loss of life. Depression has been associated with the disruption of neuroplasticity that is induced by stress, lifestyle challenges, illness etc4. Changes in plasticity in different regions of the brain have been associated with depression – the hippocampus region was shown to have reduced long-term potentiation (LTP) and increase long term depression (LTD) in people with depression4. Plasticity changes in the prefrontal cortex and the amygdala were also reported to be associated with depression symptoms. While therapies are available for depression including SSRI (selective serotonin reuptake inhibitors), they are associated with significant side effects especially after long term use. Additionally, current anti-depressants have been shown to have significant withdrawal issues that can exacerbate depression. Due to these factors, there is a large unmet clinical need for improve anti-depressant therapies that are safe for long term use.

 Psychoplastogens are compounds that can induce rapid changes in neuronal plasticity that ultimately result in positive changes in behavior and response to stress. Several natural psychoplastogens including naturally occurring compounds, such as psilocybin, N,N-dimethyltryptamine, and 7,8-dihydroxyflavone have been isolated from mushrooms, cacti and other plants5. While these psychoplastogens have been shown to induces changes in neuroplasticity, many have been shown to have undesired side effects including hallucinations and, in some cases, have been shown to aggravate conditions such as schizophrenia5. Until recently, the development of psychoplastogens for the treatment and management of neuropsychological conditions was limited. However, in 2019, the FDA approved the first psychedelic drug to treat depression (esketamine)6, and several clinical trials that evaluate psychedelic compounds including MDMA (commonly known as ecstasy) and psilocybin are either ongoing or have recently concluded7.

These developments have reignited industry interest in evaluating psychoplastogens as treatments for neuropsychological diseases. Indeed, one of the new biotech players in the space, Delix Therapeutics, was co-founded by David Olson, an academic at the University of California, Irvine whose research focused on psychoplastogens with reduced hallucinogenic effects8. Delix is one of a few new biotechs in the space – others include ATAI Life Sciences and Gilgamesh Pharmaceuticals that are working on new psychoplastogens or new versions of existing psychedelics that have the ability to induce changes in neuroplasticity without the side effects of hallucinations or withdrawal symptoms8. The new generation of psychoplastogens have not yet been tested in humans but are expected to move into clinical trials soon. The hope is that the development of these new therapies will be able to help millions of people who suffer from depression and other neuropsychological conditions regain their wellbeing and enjoy a good quality of life.

References:

1https://www.frontiersin.org/articles/10.3389/fncel.2019.00066/full

2https://www.news-medical.net/life-sciences/What-is-Neuronal-Plasticity-and-Why-Is-It-Important.aspx

3https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944261/

4https://www.hindawi.com/journals/np/2017/6871089/

5https://www.mdpi.com/1420-3049/25/5/1172

6https://endpts.com/the-mind-blowing-rd-renaissance-in-psychedelic-meds-finds-a-home-at-johns-hopkins/

7https://www.nature.com/articles/d41586-021-00187-9

8https://endpts.com/a-new-psychedelics-player-emerges-to-treat-mental-health-disorders-minus-the-hallucinogenic-effects/