What's new

What's New

15 | Mar | 2023


Nanobodies are unique single domain antibodies that are expressed in camels, alpacas, llamas and other camelid animals. These tiny antibodies are about 10% the size of regular antibodies and have a mass of about 15 kDa and due to their small size, they were named nano-antibodies or nanobodies. They were discovered in the 1980s and were found to contain only a single variable domain of the antibody heavy chain1. The single variable domain contains an antigen binding site and is considered to be the smallest functional antibody fragment discovered so far. Nanobodies have been found to have desirable biophysical characteristics such as prolonged shelf life, resistance to heat, chemical or proteolytic degradation, effective tissue penetration and low immunogenicity2. Additionally, there are reports that nanobodies can refold into functional conformations after heat denaturation2 but this is currently under debate. Nanobodies have a unique structure where they form a finger-shaped loop that can penetrate the antigen binding site or active site of an enzyme target. In contrast, conventional antibodies form a cup shaped structure that may not bind directly to the target site on the antigen3. Initially, scientists developed nanobodies by immunizing llamas and other camelids and then screening their sera for target nanobodies. However, this method had limited success and was very expensive and time consuming. Additionally, access to large animal facilities for immunization and collection were limited. However, scientists at Harvard developed a yeast-based system to express nanobodies thus avoiding the need to immunize large animals4. Nanobodies have relatively simple monomeric structures that are not post-translationally modified allowing for scalable expression in bacterial or yeast systems at milligram per liter levels. The low-cost manufacturing process that produces reproducible levels of nanobodies is highly desirable for therapeutic antibody manufacturing2. Due to the small size of nanobodies, they can be delivered using multiple methods including aerosols, which can help broaden patient access to the therapies.

Nanobodies received a lot of interest as a therapeutic modality when the first nanobody therapeutic was approved in 20195 by the European Medicine Agency (EMA) and FDA. Caplacizumab was the first nanobody based therapeutic that was initially developed by Ablynx, which was then acquired by Sanofi5. The FDA approved caplacizumab for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP), a rare clotting disease5. aTTP causes a disruption in the clotting cascade resulting in the formation of large multimers of the von Willebrand factor protein that bind to platelets forming clots or thrombi that result in emboli and other complications. Caplacizumab binds to the von Willebrand factor protein at a specific site and prevents the formation of the large multimers that cause clots and emboli.

The global SARS-CoV2 pandemic has triggered frantic efforts to find a cure for the disease that has resulted in millions of deaths. Currently, three monoclonal antibody based therapeutic regimens have received Emergency Use Authorizations (EUA) from the FDA for use in mild to moderate cases6. However, these antibodies are effective during a short time window in the early stages of infection and administering the therapies have significant logistical challenges7. Therefore, there is a strong clinical need for effective therapies that can be manufactured rapidly in a cost-effective manner and have optimal stability with minimal lot to lot variation. Nanobodies are a viable option as a therapeutic for SARS-CoV2 and recently, a group of US and EU scientists developed nanobodies targeting the SARS-CoV2 spike protein8. They developed a biparatopic nanobody which recognizes two distinct regions in the spike protein that can be delivered using an aerosol directly into the lungs of infected patients to inhibit viral infection. The researchers engineered nanobodies that neutralized the spike protein binding to the cell receptor via a unique mechanism. The nanobodies bound to the inactive SARS-CoV2 spike protein to induce a conformational change that resulted in premature inactivation of the spike protein. In other words, the nanobodies caused a change in the spike protein structure which prevented the virus from binding to and infecting cells8. These results suggest that engineered nanobodies could be the therapeutic answer to manage the pandemic that has ravaged the world.










15 | Feb | 2023


Artificial Intelligence or AI has been touted as the groundbreaking approach to more efficient and cost-effective drug discovery. At its core, AI is a combination of several computational techniques that require programming and training, that can rapidly analyze enormous datasets. However, it is important to note that output from an AI platform will only be good as the algorithms and size and quality of input datasets1. Given that the drug development consists of several steps where each step generates large amounts of data, AI applications will help streamline the process and potentially cut time and costs. An added benefit is that AI will help minimize human inefficiency and errors that will help standardize the drug design, screening and validation process2 and AI can also help weed out drug candidates that are likely going to fail in downstream validation. This allows drug developers to focus on viable candidates that have a higher likelihood of success.

Recently, several startup companies have been developing cutting-edge AI methods and the pharmaceutical industry has been quick to leverage the available expertise via large dollar collaborations. One example is Sanofi who has announced a couple of large AI collaborations – in January 2022, Sanofi expanded a partnership with Exscientia to deliver up to 15 new targets in oncology and immunology for an upfront payment of $100 million3. If the candidate search shows clinical and commercial success, the deal could net Exscientia up to $5.2 billion. Additionally, Sanofi recently signed a deal with Atomwise, an AI company that has a proprietary platform for structure-based drug design, for $20 million upfront and up to $1.2 billion if the program shows success4. Not be outdone, Merck has teamed up with Absci in a deal that is valued up to $610 million to use their Integrated Drug Creation platform to identify 3 disease targets along with therapies for those targets5. Amgen has teamed up with Generate Biomedicines, an AI company that is generating a lot of interest, to identify multi-specific drugs across various disease indications6. Similar to other AI deals, Amgen has committed to paying $50 million upfront and up to $1.9 billion in milestones if the targets achieve success6.

These collaborations seem to follow a similar pattern where pharma companies essentially fund small AI companies to refine and test their programs upfront with the promise of huge payouts if the AI platform generates viable candidates that show clinical and commercial success. This suggests that AI driven drug discovery is considered to be in its early days, especially since there are no data as yet to show that AI methods do result in more effective and cheaper drugs. Indeed, a poll by a pharma trade magazine showed that about a third of respondents believe that AI will peak after about a decade7.

One area where AI seems to have a more widespread effect is diagnostics. The most commonly used diagnostic method is pathology based where tissue samples are histologically analyzed manually. Manual diagnosis is time consuming and introduces human error due to subjective analysis of specific tissue sections. AI based methods have the potential to speed up accurate diagnosis, reduce human error and provide insights into disease biology8. Digital pathology has made significant strides in recent years and complete digital pathology workflow systems that have been approved by the FDA are available9. Advances in digital pathology-based diagnoses have been seen in the cancer space and this has helped pathologists provide more accurate diagnoses as well as assess biomarker expression for targeted therapies9. It is evident that AI will continue to advance precise diagnostics in order to support targeted therapies and precision medicine.











31 | Jan | 2023


Mouse models have been extensively used to study the onset and development of neuronal diseases, and evaluate response to therapies. These models of neurodegenerative disease have been generated using multiple approaches including genetic engineering, pharmacological stimuli and seeding of disease cell lysates1.For example, several types of transgenic models of Alzheimer’s disease (AD) that focus on beta-amyloid (APP) or tau pathologies have been developed to study the pathophysiology of Alzheimer’s disease as well as other types of dementia. Parkinson’s disease model can be broadly segmented into 2 types – 1) pharmacological models where chemicals such as 6-hydroxydopamine are used to damage and destroy dopaminergic neurons or 2) transgenic models that have mutations in genes that are known to be associated with Parkinson’s disease. Despite the decades of work and billions of dollars spent on these models, it is evident that mouse models of neurodegenerative diseases are not fully representative of the disease state and do not recapitulate the overall disease phenotype1. There are several critical differences between human disease and modeling the disease in mice that limits the translatability of mouse model data to human patients such as the difference in biomarker endpoints and the physiological differences between mouse and human brains. The lack of physiologically relevant animal models that recapitulate an acceptable level of disease pathophysiology is one of the main reasons that no curative therapies have been developed for neurodegenerative diseases such as AD, Parkinson’s or ALS.

Despite the challenges, mouse models are critical tools for preclinical drug development, so, in an effort to improve the translatability, scientists are developing chimeric mouse models. Chimeric mice, as the name suggests, are developed by transplanting human cells into the mouse brain. The transplanted human cells are typically derived from induced pluripotent stem cells (iPSCs) that can be genetically modified if required2. A 2019 study from a Belgium research group demonstrated that ES cell derived cortical pyramidal neurons when injected into the mouse brain cortex with EGTA (to facilitate integration) were able to not only integrate but also migrate through the mouse cortex while remaining viable and functional2. A percentage of transplanted neurons were shown to respond to visual stimuli. This finding suggests that transplanting stem cell derived neurons into mouse brains could be a model to study neuronal plasticity and could even be a cell therapy-based strategy to reverse brain damage2.

There is a growing body of work on the development of chimeric mice to model specific disease states including Alzheimer’s disease. One of the earliest reports on the development of chimeric AD models was in 2017 where researchers transplanted iPSC derived neurons into the brains of AD mice3. Unfortunately, this strategy had limited success as the neurons died before the development of neurofibrillary tangles. A recent paper advanced the work by transplanting astrocytes derived from iPSCs of AD patients into a transgenic Alzheimer’s disease model4. The iPSC derived astrocytes expressed either ApoE3 that is not associated with AD or ApoE4 that is strongly associated with late onset of AD. The transplanted astrocytes integrated into the mouse brain and were shown to acquire human astrocyte specific morphologies that are different from rodent astrocytes. More interestingly, the transplanted human astrocytes responded to the A-beta deposits in the mouse AD model where some of the astrocytes became hypertrophic and others atrophied. Astrocyte hypertrophy is considered to be a defense against AD pathology while atrophy is a loss of function associated with aging and neurodegenerative disease. The presence of both hypertrophy and atrophy in the AD mouse brain suggests that a chimeric model could provide valuable insights into early AD development and this information is critical to develop therapies that can significantly delay, halt or even reverse early AD development. While these are early days, the data suggests that chimeric mice might be the next generation of mouse models to study the onset and development of complex neurodegenerative diseases such as Alzheimer’s disease.






03 | Jan | 2023


Animal models have been widely used to study neurological diseases including neurodegenerative, developmental, and rare CNS disorders. However, commonly used rodent models give limited information on physiology and disease pathophysiology, as there are significant differences between rodent and human brains. The human brain has over a thousand times more neurons than the mouse brain, and single cell RNA sequencing has shown that there are gene expression differences1.While several cell types are common between human and rodents, certain cell types are missing in rodents such as the outer radial glial cells that are progenitor cells for neocortex development2. Non-human primates are an alternative to rodent models as they recapitulate the structure and function of the human CNS and can also be used for behavioral studies. However, due to the COVID-19 pandemic, there has been a significant shortage of these models which has impacted the pace of biomedical research3. Not surprisingly, there is an increasing interest in developing three-dimensional brain organoids to study disease pathophysiology and test novel therapies. Brain organoids are defined as self-assembled aggregates of cells that include more than one cell type and are developed from either embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. Several studies have shown that CNS organoids are able to recapitulate structural and functional characteristics including neurogenesis, cell migration and neural circuitry4.

One of the major issues with using CNS organoids is apoptosis and necrosis in the core. Organoids rely on diffusion for gas exchange, nutrients and waste removal, which is too inefficient to maintain viable long-term culture. The study of neurodegenerative diseases typically requires long term cultures to study disease progression, so it is essential to improve the duration of viable cultures. One solution is to introduce vasculature in the grafting organoids in adult mouse brains via the recruitment of mouse endothelial cells. The development of vascular organoids (vOrganoids) is another method to introducing vasculature in organoid culture where organoids are co-cultured with human vascular endothelial cells (HUVECs). The co-culture approach was shown to induce the formation of a tubular network of blood vessels allowing the culture of organoids for up to 200 days5.

CNS organoids can be generated using two different methods: unguided and guided6. Unguided development of CNS organoids depends on the intrinsic self-organization capabilities of pluripotent stem cells to form defined cellular structures without the aid of any external factors. One of the drawbacks with unguided organoid generation is the variation in cell populations and size of the organoids. Guided methods introduce more control in organoid assembly via exposure to specific patterning factors at specific times. The presence of specific factors triggers specific signaling pathways to generate organoids of specific lineages. For example, the use of a Smad inhibitor shifts the organoids away from mesoderm and endoderm lineages and towards ectoderm lineages7.

The use of iPS cells to generate CNS organoids has broadened the applications of CNS organoids as patient derived cells can be used to generate disease specific organoids. Brain organoids are also used to study neuronal development and diseases associated with neurodevelopment such as microcephaly, macrocephaly, and autism spectrum disorders. Another big area of interest is the study of signaling pathways associated with neuropsychiatric diseases4, so that more targeted and efficacious therapies can be developed. One of the largest and well-funded neuroscience research areas is neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease and the development of disease specific organoids that can be maintained in long term cultures will be very useful to test therapies to slow or reverse neurodegeneration.









11 | Oct | 2022


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.










15 | Sep | 2022


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.







18 | Aug | 2022


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.








28 | Jul | 2022


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.







05 | Jul | 2022


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.









09 | Jun | 2022


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.