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/

01 | Feb | 2022

One of the largest unmet clinical needs are disease modifying therapies for Alzheimer’s disease (AD), which is the most common neurodegenerative disease and is responsible for about 50% of dementia cases1. More than 6 million Americans live with AD and 1 in 3 seniors will die from AD or another form of dementia1. Currently, there are four approved therapies to treat symptoms related to cognition – Donepezil, Rivastigmine and Galantamine are cholinesterase inhibitors while Memantine is a glutamate regulator. These therapies do not address the underlying disease pathophysiology so finding new AD therapies is an area of active research. It is estimated that the NIH spend on AD is $3.1 billion¹ and there are several foundations that support drug development studies and patient care. However, the failure rate of Alzheimer’s disease clinical trials is over 99%² where the drug target is primarily β-amyloid or tau protein. Therefore, researchers are searching for new drug targets in cellular processes implicated in plaque or tangle formation seen in AD.

A lab at the Albert Einstein College of Medicine has taken a new approach to developing a drug that targets autophagy, which removes cellular waste and protein buildup. Autophagy has been an area of interest in neurodegenerative diseases and the researchers at Albert Einstein College of Medicine focused on chaperone-mediated autophagy, where chaperone proteins in the cell select specific proteins for degradation. They studied the importance of chaperone-mediated autophagy in the brain and found that when the process was disrupted, then insoluble protein aggregates accumulated in neurons³. The study also identified the critical drug target – LAMP2A, a lysosome associated protein that plays a critical role in chaperone-mediated autophagy. When LAMP2A was disrupted in mouse brain, the animal developed Alzheimer’s disease like symptoms including limited mobility and memory impairment due to inhibition of chaperone-mediated autophagy⁴. Interestingly, single cell RNA sequencing of AD brain tissues also showed inhibition of chaperone-mediated autophagy which increased with disease severity⁵. This key finding where a mouse model recapitulated the human disease state suggests that preclinical models can be very useful in understanding the role of chaperone-mediated autophagy in AD development and progression.

The next step in the study was the synthesis of an activator of chaperone-mediated autophagy called CA. When this drug was administered orally to mice with abnormalities in tau and β-amyloid or tau alone, it significantly reduced tau protein levels and β-amyloid aggregates and the animals had improvement in cognition, depression and anxiety over 4-6 months of drug dosing⁶. Another positive outcome of the study was that oral dosing of the drug did not cause adverse events in mice. Instead, the drug induced a reduction in gliosis which is the inflammation and damage in cells surrounding the neurons. Given these promising results, the lead investigators have founded a company called Selphagy Therapeutics to develop the CA drug and next-generation therapies that target chaperone-mediated autophagy and move these therapies into the clinic.

While it is not clear how these preclinical findings will translate to response in human patients, new therapies like CA that target disease relevant cellular processes have the potential to delay or even reverse neurodegeneration and neuronal cell death. Studies like these bring hope to the millions of patients with dementia and their families.

References:

¹https://www.alz.org

²https://www.beingpatient.com/alzheimers-drug-trials-failure-rate/

³https://www.fiercebiotech.com/research/treating-alzheimer-s-disease-by-invigorating-cell-s-specialized-garbage-cleaning-system

⁴https://www.sciencedirect.com/science/article/abs/pii/S0092867421003792

⁵https://www.nature.com/articles/s41467-021-22501-9#citeas

⁶https://www.sciencedaily.com/releases/2021/04/210422150402.htm

05 | Jan | 2022

RNA-based therapies are a fast-growing class of drugs that utilize the cellular translational machinery and have the potential to change treatment paradigms for several diseases. Current therapeutic strategies include RNA aptamers that bind receptors to inhibit downstream signaling, RNA interference using either siRNA (small interfering RNA) or miRNA (microRNA) that bind to the endogenous mRNA sequences to promote degradation and mRNA-based therapies that impact translation1¹. Several RNA based therapies have been approved by regulatory agencies, and some of the first RNA therapies approved in 2016 targeted rare diseases including spinal muscular atrophy and Duchenne muscular dystrophy1. A Several RNA therapies are currently being tested in various phase I, II or III clinical trials. Notably, two mRNA vaccines from BioNTech/Pfizer and Moderna have received Emergency Use Authorization for COVID-19.

A new type of RNA, long noncoding RNA (lncRNA), is an area of active research to develop novel RNA therapies but the challenge is that these circular RNAs are poorly recognized by ribosomes. Recently, a new RNA type called endless RNA (eRNA) that combines a circular structure that can be easily translated². aA new biotech company, Laronde Bio, was recently launched to develop eRNA based therapies. Endless RNAs contain an internal ribosomal entry site or IRES that allows ribosomes to bind to the circular RNA for translation and due to the circular structure, de novo protein synthesis is consistently going on in a loop. The circular structure makes the eRNA highly stable as RNases and other degradation machinery do not have a loose end to bind to start the RNA breakdown process. Since the eRNA is made with regular unmodified nucleotides it is likely not going to trigger an innate immune response so the residence time in the cell is expected to be long. This will allow a larger therapeutic efficacy window and allow repeated dosing to further increase the duration of therapeutic protein expression. The relatively simple structure of eRNA is likely going to support scalable manufacturing in a cost-effective manner that are highly desirable for large molecule therapies.

One of the key questions is efficient delivery of eRNA in vivo. Currently, RNA therapies are most effectively delivered via direct injection into the target organ. For example, Spinraza®, an antisense oligonucleotide therapy for spinal muscular atrophy is directly injected into the cerebrospinal fluid so that it reaches the motor neurons in the spinal cord. Another option is to link the RNA therapy to lipids, peptides, antibodies or sugar moieties to facilitate cell uptake³. With the advances in delivery of nucleic acid therapeutics, there is a good chance that the most efficient delivery method for eRNA will be identified. For now, Laronde Bio has raised $50M in financing from Flagship Pioneering and has set a lofty goal of producing 100 eRNA based medicines in 10 years⁴. Currently, the target diseases have not been announced but will fall into the broad areas of hematology, oncology, neurology, immunology and inflammation. One of the key advantages with eRNA is that each therapy does not need to be built from scratch – the eRNA backbone is modifiable and coding regions of various therapeutic genes can be inserted and swapped with ease. It remains to be seen if there is a size limitation on the expression cassette but for now, eRNA has the potential to fundamentally change RNA based therapies. If this technology shows clinical value, it has the potential to fundamentally alter the timelines and costs associated with drug development.

References:

¹https://www.frontiersin.org/articles/10.3389/fbioe.2021.628137/full

²2. https://www.prnewswire.com/news-releases/flagship-pioneering-unveils-laronde-to-advance-endless-rna—a-new-class-of-programmable-medicines-capable-of-expressing-therapeutic-proteins-inside-the-body-301287088.html

³Roberts, T.C., Langer, R. & Wood, M.J.A. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov 19, 673–694 (2020).

⁴https://www.biopharmadive.com/news/laronde-flagship-launch-endless-rna-erna/599841/

01 | Dec | 2021

CRISPR technology 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 the landmark publication that described the complete CRISPR-Cas9 system was published in the journal Science¹. Since the first report, the number of publications has been steadily increasing – over 17,000 papers on CRISPR were published in 2018² and the numbers continue to increase. This is clear evidence that CRISPR is being widely adopted in research and development applications. Another breakthrough in CRISPR technology was the launch of the first clinical trials. Several early-stage clinical trials have been launched and completed in various disease areas including cancer, blood diseases, and rare diseases³.

The first cancer clinical trial using CRISPR was performed in China in 2016 where non-small cell lung cancer patients was injected with PD-1 edited T cells in a phase I trial to evaluate the safety of the therapy. The results from this study were published in 2020⁴, and overall, the edited T-cells were well tolerated and the off-target effects of CRISPR gene editing were infrequently found. The first clinical trial in the US used a more complicated approach where CRISPR was used to knockout PD-1 and endogenous T-cell receptor and introduce an engineered T-cell receptor in T-cells. These engineered T-cells were introduced into 3 patients – 2 with advanced refractory myeloma and one with metastatic sarcoma). The study found that the multiplex CRISPR-edited T-cells showed an acceptable safety profile and the engineered T-cells were detectable in the patients’ bone marrow and tumors⁵.

Correcting underlying issues in blood diseases has been an active area of CRISPR research. The first report of CRISPR-based therapies was a study done in 2019 on beta-thalassemia patients where edited hematopoietic stem cells from healthy donors were successfully introduced into patients. The first patient with sickle cell disease, Victoria Gray, was also treated in 2019 with edited cells and the results have shown that this approach is successful in increasing hemoglobin levels to close to normal and more importantly, the engineered cells are detectable in the bone marrow suggesting that infusion of CRISPR-edited cells could have a long-term effect in patients with sickle cell disease or beta-thalassemia³. More recently, scientists at UCSF, UC Berkeley and UCLA have launched an early-stage trial to test a CRISPR solution to directly correct the sickle cell mutation in the patient’s own blood cells⁶ that has the potential to be a true cure.

The first trial in an eye disease was launched last year where patients with Leber congenital amaurosis 10 (LCA10) were dosed with an adeno-associated virus (AAV) carrying the Cas9 protein and guide RNA under the control of a tissue-specific promoter so that the CRISPR system is only expressed in photoreceptor cells⁷. LCA is caused by mutations in the centrosomal protein 290 so correction of the genetic defect in the gene is hypothesized to improve vision in LCA patients. Injecting the CRISPR system directly into the eye has significant advantages since the eye is immune-privileged and contained so there is a lower risk of off-target effects outside the eye. Indeed, in mouse studies, about 10% of photoreceptor cells showed the edit and this level of correction is considered to be sufficient to improve vision. 

Based on these recent trials, it is clear that CRISPR is progressing beyond a powerful research tool and developing into a truly curative system for several diseases. Monitoring the off-target effects and adverse events in patients are critical to understand the short and long-term implications of CRISPR therapies and ensure that the system is used in an ethical way to improve patient lives and cure diseases.

References:

¹https://science.sciencemag.org/content/337/6096/816.abstract

²https://www.vox.com/2018/7/23/17594864/crispr-cas9-gene-editing

³https://www.newswise.com/articles/crispr-clinical-trials-a-2021-update

⁴Lu, Y., Xue, J., Deng, T. et al. Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat Med 26, 732–740 (2020).

⁵https://science.sciencemag.org/content/367/6481/eaba7365

⁶https://www.ucsf.edu/news/2021/03/420137/uc-consortium-launches-first-clinical-trial-using-crispr-correct-gene-defect

⁷https://ir.editasmedicine.com/news-releases/news-release-details/allergan-and-editas-medicine-announce-dosing-first-patient

08 | Nov | 2021

There has been a lot of activity and progress in the development and approval of new therapies for eye diseases in the past few years. Five of the 178 new drugs approved between October 2019 and December 2020 targeted eye diseases, and 2 lens-based devices were also approved in the same time frame¹. Currently, there are over 1,500 interventional clinical trials for various eye diseases² so the pace of new drug development does not appear to be slowing down.

An important aspect of drug development is drug delivery to ensure maximum bioavailability and efficacy. Various drug delivery methods are used for eye medications including eye drops, gels, ocular inserts, contact lenses and injections into specific parts of the eye3³. However, the unique anatomy and physiology of the eye poses challenges for effective drug delivery especially to the inner eye. While topical delivery is preferred for drugs administered to the front of the eye, more invasive intravitreal injections are typically used to deliver drugs to the inner eye. Topical delivery is the least invasive method to delivery drugs as eye drops or emulsions but the tear film dilutes and washes away the drug reducing bioavailability.

Nanoparticles have several characteristics that are suitable for delivery to the eye. The small size allows movement across various barriers and tissues and therapeutic modalities can be attached to the nanoparticles for delivery to the area of interest. The path of nanoparticle movement and distribution in the eye can be controlled to some extent via the modification of the size, charge and solubility of the nanoparticles as well as the route of administration⁴, so it is important to design the delivery strategy optimally for the drug. The physical and chemical characteristics of the nanoparticle should be carefully designed for optimal biodistribution and should be tested on the most relevant preclinical animal models prior to clinical testing. One example is the design of gold nanoparticles to penetrate the blood-retina barrier (BRB). Studies have shown that the nanoparticles have to be 20 nm in size to successfully penetrate the BRB as 100 nm particles are too large⁵. Tracking the nanoparticle after injection can be done using fluorescence if a dye is included in the nanoparticle or a reporter marker linked to the nanoparticle. The choice of a tracking marker should be carefully tested preclinically to ensure that the biodistribution of the nanoparticles is not impacted. Another characteristic that should be considered is controlled release where drugs can be released from the nanoparticle in response to specific stimuli such as pH or light⁶. This prevents drug leakage locally or into systemic circulation to reduce adverse events and increase bioavailability of the drug.

The administration route for nanoparticles is a critical component of successful drug delivery. The cornea is the outermost barrier for topical administration but penetration enhancers can be used to move nanoparticles past the cornea. Topical delivery of nanoparticles does allow diffusion through the eye to the retina but has several challenges – the nanoparticles have to penetrate through the cornea and choroid before reaching the retina and then passing through the BRB. However, a significant number of nanoparticles enters the systemic circulation at the BRB, so higher doses of nanoparticles are needed to meet the therapeutic threshold. Though invasive, targeted injections of nanoparticles can be used to deliver drugs to specific areas in the drug. Suspending nanoparticles in hydrogels or highly viscous polymers helps immobilize them and reduces clearance. Coating nanoparticles in polyethylenglycol (PEG) helps reduce immune clearance in vascularized regions of the eye.

Nanoparticles have a great deal of promise to deliver ocular drugs and there is active research ongoing to identify particle materials that have optimal characteristics to penetrate the various static and dynamic barriers in the eye to reach the area of interest. Continuously improving systems and devices that deliver novel drugs for various ocular diseases appears to be the path of the future which is looking very bright indeed.

References:

¹www.healio.com/news/ophthalmology/20201216/number-of-fda-new-molecular-entity-approvals-in-2020-similar-to-2019

²https://clinicaltrials.gov/

³Gorantla S et al. Nanocarriers for ocular drug delivery: current status and translational opportunity. RSC Adv 2020 10, 27835.

⁴Swetledge, S et al. Distribution of polymeric nanoparticles in the eye: implications in ocular disease therapy. J Nanobiotechnol 2021; 19, 10.

⁵• 5Kim JH et al. Intravenously administered gold nanoparticles pass through the blood-retinal barrier depending on the particle size, and induce no retinal toxicity. Nanotechnology. 2009; 20(50):505101.

⁶https://globalrph.com/2021/01/use-of-nanotechnology-to-deliver-topical-ophthalmic-medications/” target=”_blank

01 | Oct | 2021

Gene therapy, by definition, is the replacement of a malfunctioning or missing gene to correct a genetic disease. While several gene therapies have been approved for monogenic diseases, therapies targeting more complex polygenic diseases pose challenges. For example, if the expression of a specific ligand is downregulated in the disease state, simply increasing ligand expression via a gene vector may not be sufficient to ameliorate the disease if receptor expression is also downregulated due to low intrinsic ligand expression. In other words, correcting a single gene deficiency is likely not sufficient to reverse or improve the disease state if multiple genes are involved. Recently, groups from Cambridge University and University of Melbourne published a proof-of-concept paper¹ where they successfully demonstrated gene expression of 2 genes delivered using a single adeno-associated virus (AAV).

While a combination gene therapy approach has been published before², it has utilized individual vectors to deliver each gene. A study from George Church’s lab showed that a combination of 3 genes associated with increasing longevity – FGF21, αKlotho and soluble TGFβ receptor2 – had a positive impact on 4 age related diseases including obesity, type 2 diabetes, heart failure and renal failure. Systemic delivery of AAVs singly or in various combinations showed a positive effect on all 4 disease models. The combination of TGFβ receptor2 and FGF21 expression showed synergistic improvement of therapeutic efficacy in all 4 diseases, but the combination of FGF21 and αKlotho had a negative effect on the renal and heart failure disease models. This interesting finding suggests that designing gene combinations should be done carefully and validated in preclinical models for synergy. Nevertheless, the study by Davidsohn et al. highlight a new approach to gene therapy for polygenic diseases but this approach has some limitation including differences in expression level of the individual genes that are under the control of different promoters and concerns with viral burden.

The recent work from Khatib et al. has advanced combination gene therapy by combining expression of 2 genes in a single viral vector under the control of one promoter – BDNF or brain derived neurotrophic factor and its receptor TrkB. The two open reading frames are separated by a viral 2A peptide that is cleaved by the host cell machinery resulting in the expression of both genes in the same cell and at similar expression levels. This novel viral vector was tested in 2 animal models of disease – experimental glaucoma and tauopathy. Glaucoma is an optic neuropathy that causes optic nerve damage so the glaucoma phenotype was induced via injury to the optic nerve. The increased expression of both BDNF and TrkB was shown to improve axonal transport along the optic nerve compared to single expression of each gene and there was improved vision suggesting that this combination gene therapy could potentially restore vision loss due to glaucoma.

There have been reports that showed correlation in amyloid-related pathology in the eye and brain so the effect of improved axonal transport along the optic nerve was tested in the P301S mouse model of tauopathy measured by measurement of improvement of short-term and long-term memory. The study showed moderate increase in short-term memory after AAV administration but no significant change in long-term memory.

It is clear that combination gene therapies have the potential to improve gene therapy and there are signs of pharma interest in this area. The group from Cambridge University spun out a company called Quethera that is focused on the next generation of combination gene therapies. Astellas recently acquired Quethera in a deal valued at up to $109M^{3 4}suggesting that combination gene therapies for polygenic diseases is the next frontier of gene therapy development.

References:

¹Khatib et al. Receptor-ligand supplementation via a self-cleaving 2A peptide–based gene therapy promotes CNS axonal transport with functional recovery Science Advances 31 Mar 2021: Vol. 7, no. 14, eabd2590.

²Davidsohn et al. A single combination gene therapy treats multiple age-related diseases. Proceedings of the National Academy of Sciences Nov 2019, 116 (47) 23505-23511.

³https://newsroom.astellas.us/2018-08-10-Astellas-Announces-Acquisition-of-Quethera/

⁴https://www.fiercebiotech.com/research/astellas-gene-therapy-repairs-damage-neurodegenerative-disease-models” target=”_blank