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DNA repair discoveries hold promise for new approaches to cancer treatment

by NYU Langone Health

DNA can be damaged by toxins, radiation, or even normal cell division, but human cells must continually fix DNA breaks to survive. In cells that cannot repair DNA effectively, changes (mutations) can occur that lead to cancer.

Most cells rely on a system called homologous recombination or HR, which uses proteins called BRCA1 and BRCA2 for accurate DNA repair. Those born with a malfunctioning BRCA gene, however, often develop breast and ovarian cancers , with BRCA mutations and related HR problems recently found to occur in pancreatic and prostate cancer as well.

For this reason, identifying patients with "HR-deficient" cancers has become a priority in the field, in part because such cancer cells are vulnerable to targeted therapies that break their DNA. To find patients with HR deficiency, standard lab tests look for " scars " in the DNA of cancer cells, which happen when sloppy, back-up repair processes are used instead of HR to create specific mutation patterns.

While accurate scar diagnosis enables more tailored treatment, researchers have been puzzled by the subtly of the scars found in HR-deficient cancers. Such scars create very small typos in the DNA code (sequence), which are invisible under the microscope. However, HR-deficient cells show dramatic structural rearrangements in much larger DNA structures called chromosomes that are visible by microscopy.

To tackle this paradox, Marcin Imieliński, MD, Ph.D., at NYU Langone Health's Perlmutter Cancer Center, and Simon Powell, MD, Ph.D., at Memorial Sloan Kettering Cancer Center (MSKCC), applied "genome graph" techniques developed in Imieliński's lab to detect massive structural DNA changes that rearrange, copy, and delete huge sections of chromosomes.

Published online August 16 in the journal Nature, their study also analyzed DNA molecules a hundred times longer than those normally measured in cancer analyses.

Applying these methods, the research team identified "reciprocal pairs," a new scar type seen in HR deficiency. By analyzing thousands of cancer genomes, the research team showed that when HR fails, reciprocal pair scars create specific chromosomal changes visible by microscope and that better explain the biology of HR-deficient cells.

"The long molecules tell us that these scars come from two backup repair mechanisms—homology-independent replication restart and single strand annealing—that may keep HR-deficient cancer cells alive," says Imieliński, director of Cancer Genomics at Perlmutter Cancer Center, and an attending pathologist at NYU Langone. "Blocking the mechanisms may represent new ways to treat these cancers."

The study authors note that their new techniques require the use of a technology called whole genome sequencing (WGS), but that WGS costs are falling. The researchers say it may soon be practical to use their approach to find more HR-deficient patients and match them with targeted therapies.

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A new technique for correcting disease-causing mutations

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Gene editing, or purposefully changing a gene’s DNA sequence, is a powerful tool for studying how mutations cause disease, and for making changes in an individual’s DNA for therapeutic purposes. A novel method of gene editing that can be used for both purposes has now been developed by a team led by Guoping Feng , the James W. (1963) and Patricia T. Poitras Professor in Brain and Cognitive Sciences at MIT.

“This technical advance can accelerate the production of disease models in animals and, critically, opens up a brand-new methodology for correcting disease-causing mutations,” says Feng, who is also a member of the Broad Institute of Harvard and MIT and the associate director of the McGovern Institute for Brain Research at MIT. The new findings were published online May 26 in the journal Cell .

Genetic models of disease

A major goal of the Feng lab is to precisely define what goes wrong in neurodevelopmental and neuropsychiatric disorders by engineering animal models that carry the gene mutations that cause these disorders in humans. New models can be generated by injecting embryos with gene editing tools, along with a piece of DNA carrying the desired mutation.

In one such method, the gene editing tool CRISPR is programmed to cut a targeted gene, thereby activating natural DNA mechanisms that “repair” the broken gene with the injected template DNA. The engineered cells are then used to generate offspring capable of passing the genetic change on to further generations, creating a stable genetic line in which the disease, and therapies, are tested.

Although CRISPR has accelerated the process of generating such disease models, the process can still take months or years. Reasons for the inefficiency are that many treated cells do not undergo the desired DNA sequence change at all, and the change only occurs on one of the two gene copies (for most genes, each cell contains two versions, one from the father and one from the mother).

In an effort to increase the efficiency of the gene editing process, the Feng lab team initially hypothesized that adding a DNA repair protein called RAD51 to a standard mixture of CRISPR gene editing tools would increase the chances that a cell (in this case a fertilized mouse egg, or one-cell embryo) would undergo the desired genetic change.

As a test case, they measured the rate at which they were able to insert (“knock-in”) a mutation in the gene Chd2 that is associated with autism. The overall proportion of embryos that were correctly edited remained unchanged, but to their surprise, a significantly higher percentage carried the desired gene edit on both chromosomes. Tests with a different gene yielded the same unexpected outcome.

“Editing of both chromosomes simultaneously is normally very uncommon,” explains postdoc Jonathan Wilde. “The high rate of editing seen with RAD51 was really striking, and what started as a simple attempt to make mutant Chd2 mice quickly turned into a much bigger project focused on RAD51 and its applications in genome editing,” says Wilde, who co-authored the Cell paper with research scientist Tomomi Aida.

A molecular copy machine

The Feng lab team next set out to understand the mechanism by which RAD51 enhances gene editing. They hypothesized that RAD51 engages a process called interhomolog repair (IHR), whereby a DNA break on one chromosome is repaired using the second copy of the chromosome (from the other parent) as the template.

To test this, they injected mouse embryos with RAD51 and CRISPR but left out the template DNA. They programmed CRISPR to cut only the gene sequence on one of the chromosomes, and then tested whether it was repaired to match the sequence on the uncut chromosome. For this experiment, they had to use mice in which the sequences on the maternal and paternal chromosomes were different.

They found that control embryos injected with CRISPR alone rarely showed IHR repair. However, addition of RAD51 significantly increased the number of embryos in which the CRISPR-targeted gene was edited to match the uncut chromosome.

“Previous studies of IHR found that it is incredibly inefficient in most cells,” says Wilde. “Our finding that it occurs much more readily in embryonic cells and can be enhanced by RAD51 suggest that a deeper understanding of what makes the embryo permissive to this type of DNA repair could help us design safer and more efficient gene therapies.”

A new way to correct disease-causing mutations          

Standard gene therapy strategies that rely on injecting a corrective piece of DNA to serve as a template for repairing the mutation engage a process called homology-directed repair (HDR).

“HDR-based strategies still suffer from low efficiency and carry the risk of unwanted integration of donor DNA throughout the genome,” explains Feng. “IHR has the potential to overcome these problems because it relies upon natural cellular pathways and the patient’s own normal chromosome for correction of the deleterious mutation.”

Feng’s team went on to identify additional DNA repair-associated proteins that can stimulate IHR, including several that not only promote high levels of IHR, but also repress errors in the DNA repair process. Additional experiments that allowed the team to examine the genomic features of IHR events gave deeper insight into the mechanism of IHR and suggested ways that the technique can be used to make gene therapies safer.

“While there is still a great deal to learn about this new application of IHR, our findings are the foundation for a new gene therapy approach that could help solve some of the big problems with current approaches,” says Aida.

This study was supported by the Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT, the Poitras Center for Psychiatric Disorders Research at MIT , an NIH/NIMH Conte Center Grant, and the NIH Office of the Director.

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January 22, 2024 | Taylor Graves - UConn School of Pharmacy

CRISPR and Other New Technologies Open Doors for Drug Development, but Which Diseases get Prioritized?

It comes down to money and science

Andriy Onufriyenko/Moment via Getty Images

Prescription drugs and vaccines revolutionized health care, dramatically decreasing death from disease and improving quality of life across the globe. But how do researchers, universities and hospitals, and the pharmaceutical industry decide which diseases to pursue developing drugs for?

In  my work  as director of the  Health Outcomes, Policy, and Evidence Synthesis  group at the University of Connecticut School of Pharmacy, I assess the effectiveness and safety of different treatment options to help clinicians and patients make informed decisions. My colleagues and I study ways to create new drug molecules, deliver them into the body and improve their effectiveness while reducing their potential harms. Several factors determine which avenues of drug discovery that people in research and pharmaceutical companies focus on.

Funding drives research decisions

Research funding amplifies the pace of scientific discovery needed to create new treatments. Historically,  major supporters of research  like the National Institutes of Health, pharmaceutical industry and private foundations funded studies on the most common conditions, like heart disease, diabetes and mental health disorders. A  breakthrough therapy  would help millions of people, and a small markup per dose would generate hefty profits.

As a consequence, research on rare diseases was not well-funded for decades because it would help fewer people and the costs of each dose had to be very high to turn a profit. Of the  more than 7,000 known rare diseases , defined as  fewer than 200,000 people affected  in the U.S.,  only 34 had a therapy approved  by the Food and Drug Administration before 1983.

The passage of the  Orphan Drug Act  changed this trend by offering tax credits, research incentives and prolonged patent lives for companies actively developing drugs for rare diseases. From 1983 to 2019,  724 drugs  were approved for rare diseases.

Emerging social issues or opportunities can significantly affect funding available to develop drugs for certain diseases. When COVID-19 raged across the world, funding from  Operation Warp Speed  led to vaccine development in record time. Public awareness campaigns such as the  ALS ice bucket challenge  can also directly raise money for research. This viral social media campaign provided 237 scientists  nearly US$90 million  in research funding from 2014 to 2018, which led to the discovery of five genes connected to amyotrophic lateral sclerosis, commonly called Lou Gehrig’s disease, and new clinical trials.

How science approaches drug development

To create breakthrough treatments, researchers need a basic understanding of what disease processes they need to enhance or block. This requires developing  cell and   animal models  that can simulate human biology.

It can  take many years  to vet potential treatments and develop the finished drug product ready for testing in people. Once scientists identify a potential biological target for a drug, they use  high-throughput screening  to rapidly assess hundreds of chemical compounds that may have a desired effect on the target. They then modify the most promising compounds to enhance their effects or reduce their toxicity.

When these compounds have lackluster results in the lab, companies are likely to  halt development  if the estimated potential revenue from the drug is less than the estimated cost to improve the treatments. Companies can charge more money for drugs that  dramatically reduce deaths or disability  than for those that only reduce symptoms. And researchers are more likely to continue working on drugs that have a greater potential to help patients. In order to obtain FDA approval, companies ultimately need to show that the drug causes more benefits for patients than harms.

Sometimes, researchers know a lot about a disease, but available technology is insufficient to produce a successful drug. For a long time, scientists knew that  sickle cell disease  results from a defective gene that leads cells in the bone marrow to produce poorly formed red blood cells, causing severe pain and blood clots. Scientists lacked a way to fix the issue or to work around it with existing methods.

However, in the early 1990s, basic scientists discovered that bacterial cells have a mechanism to  identify and edit DNA . With that model, researchers began painstaking work developing a  technology called CRISPR  to identify and edit genetic sequences in human DNA.

The technology finally progressed to the point where scientists were able to successfully target the problematic gene in patients with sickle cell and edit it to produce normally functioning red blood cells. In December 2023,  Casgevy became the first CRISPR-based drug  approved by the FDA.

Sickle cell disease made a great target for this technology because it was caused by a single genetic issue. It was also an attractive disease to focus on because it affects around 100,000 people in the U.S. and is  costly to society , causing many hospitalizations and lost days of work. It also  disproportionately affects Black Americans , a population that has been  underrepresented in medical research .

Real-world drug development

To put all these pieces of drug development into perspective, consider the  leading cause of death in the U.S. : cardiovascular disease. Even though there are several drug options available for this condition, there is an ongoing need for more effective and less toxic drugs that reduce the risk of heart attacks and strokes.

In 1989, epidemiologists found that patients with  higher levels of bad, or LDL, cholesterol  had more heart attacks and strokes than those with lower levels. Currently,  86 million American adults  have elevated cholesterol levels that can be treated with drugs, like the popular statins Lipitor (atorvastatin) or Crestor (rosuvastatin). However,  statins alone  cannot get everyone to their cholesterol goals, and many patients develop unwanted symptoms limiting the dose they can receive.

So scientists developed models to understand how LDL cholesterol is created in and removed from the body. They found that LDL receptors in the liver removed bad cholesterol from the blood, but a  protein called PCSK9  prematurely destroys them, boosting bad cholesterol levels in the blood. This led to the development of the drugs  Repathy (evolocumab) and Praluent (alirocumab)  that bind to PCSK9 and stop it from working. Another drug,  Leqvio (inclisiran) , blocks the genetic material coding for PCSK9.

Researchers are also developing a  CRISPR-based method  to more effectively treat the disease.

The future of drug developmen t

Drug development is driven by the priorities of their funders, be it governments, foundations or the pharmaceutical industry.

Based on the market, companies and researchers tend to study highly prevalent diseases with devastating societal consequences, such as  Alzheimer’s disease  and  opioid use disorder . But the work of advocacy groups and foundations can enhance research funding for other specific diseases and conditions. Policies like the Orphan Drug Act also create successful incentives to discover treatments for rare diseases.

However, in 2021, 51% of drug discovery spending in the U.S. was directed at  only 2% of the population. . How to strike a balance between providing incentives to develop  miracle drug therapies  for a few people at the expense of the many is a question researchers and policymakers are still grappling with.

Author: C. Michael White, Distinguished Professor of Pharmacy Practice, University of Connecticut.

Originally published in The Conversation.

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Mapping Study Yields Novel Insights into DNA-Protein Connection, Paving Way for Researchers to Target New Treatments

Results will help researchers “connect the dots” between DNA, proteins, and disease; could shed light on health risks and health disparities

A new genetic mapping study led by researchers at the Johns Hopkins Bloomberg School of Public Health traces links between DNA variations and thousands of blood proteins in two large and distinct populations. The results should help researchers better understand the molecular causes of diseases and identify proteins that could be targeted to treat these diseases.

The study included more than 9,000 Americans of European or African ancestry, and generated maps of DNA-to-protein links for both groups. The study is thought to be the first of its kind to include two large and ancestrally distinct population cohorts. Proteins play a critical role in cellular function, and changes in protein mechanisms—often regulated by DNA variations—can lead to disease. DNA-to-protein mapping could help explain differences in the rates of some diseases in the two groups and help researchers understand some health disparities.

The study appears May 2 in Nature Genetics .

Researchers have been mapping the molecular roots of human diseases for decades through so-called genetic mapping studies. The best known is the genome-wide association study (GWAS). A GWAS typically links variations in DNA to disease risk by analyzing the DNA of subjects—often tens or hundreds of thousands of individuals at a time—along with their history of a given disease. This uncovers statistical associations linking the disease to specific DNA variations. Missing from the GWAS picture: Most of the disease-linked DNA variants identified by GWAS analysis do not lie within protein-coding genes. Researchers therefore assumed that many—even most—disease-linked DNA variants affect proteins indirectly, by regulating one or more steps in the gene-to-protein production process, thereby altering protein levels. Linking diseases directly to proteins, researchers can better understand the roots of disease—and also identify protein targets for disease prevention and treatments.

“This relatively new kind of mapping study provides a wealth of information that will allow researchers to test for potential links of proteins on various types of health outcomes — risk of cancers, heart disease, severe COVID—and help to develop or repurpose therapeutic drugs, ” says study senior author Nilanjan Chatterjee, PhD, Bloomberg Distinguished Professor in the Department of Biostatistics at the Bloomberg School.

To demonstrate the DNA-protein mapping’s application, the researchers used it to identify an existing rheumatoid arthritis drug as a plausible new treatment for the common joint-pain disorder known as gout.

The study was a collaboration between Chatterjee’s team and the research group of Josef Coresh, MD, George W. Comstock Professor in the Bloomberg School’s Department of Epidemiology and one of the paper’s co-authors, and colleagues at several institutions.  

The analysis covered 7,213 Americans of European ancestry and 1,871 African Americans in the long-running Atherosclerosis Risk in Communities (ARIC) study, headed by Coresh; and 467 African Americans from the African American Study of Kidney Disease and Hypertension (AASK). In both of these studies, the research teams had sequenced the genomes of the participants and recorded bloodstream levels of thousands of distinct proteins.

For their mapping study, Chatterjee’s team analyzed the ARIC and AASK genomic data to identify more than two thousand common DNA variations that lie close to the genes encoding many of these proteins and correlate with the proteins’ bloodstream levels.

“The value of knowing about these DNA variants that predict certain protein levels is that we can then examine much larger GWAS datasets to see if those same DNA variants are linked to disease risks,” Chatterjee says.

Using a European-American dataset, they found that it predicted several proteins whose levels would influence the risk of gout or bloodstream levels of the gout-related chemical urate. These proteins included the interleukin 1 receptor antagonist (IL1RN) protein, which appears to lower gout risk—a finding that suggests the existing rheumatoid arthritis drug anakinra, which mimics IL1RN, as a plausible new therapy for gout.

Having data from both white and Black Americans allowed the researchers to map protein-linked DNA variants more finely than if they had been restricted to one or the other. The African-ancestry models generated in the study will allow future analyses of how different populations’ genetic backgrounds might contribute to differences in disease rates.

“We know that prostate cancer risk, for example, is higher in African American men, so in principle, one could combine prostate cancer GWAS data on African Americans with our protein data to identify proteins that contribute to elevated prostate cancer risk in that population,” Chatterjee says.

The team has made its datasets and protein prediction models publicly available online so researchers can use the resource. Chatterjee’s team and collaborators anticipate doing further studies in the ARIC and AASK cohorts, as well as in other diverse cohorts, to gather information on proteins and other factors that influence the DNA-to-disease chain of causality.

“Plasma proteome analyses in individuals of European and African ancestry identify cis-pQTLs and models for proteome-wide association studies” was co-authored by first authors Jingning Zhang and Diptavo Dutta, and by Anna Köttgen, Adrienne Tin, Pascal Schlosser, Morgan Grams, Benjamin Harvey, CKDGen Consortium, Bing Yu, Eric Boerwinkle, Josef Coresh, and Nilanjan Chatterjee.

The analysis of this project was supported by a RO1 grant from the National Human Genome Research Institute at the National Institutes of Health (1 R01 HG010480-01). Additional NIH grants supporting this research include R01 HL134320, R01 AR073178, R01 DK124399, and HL148218. The Atherosclerosis Risk in Communities study has been funded in whole or in part by the National Heart, Lung, and Blood Institute; National Institutes of Health; Department of Health and Human Services (HHSN268201700001I, HHSN268201700002I, HHSN268201700003I, HHSN268201700005I, HHSN268201700004I).

Media contacts: Carly Kempler at  [email protected]  and  Barbara Benham at  [email protected] .

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Advances in CRISPR/Cas-based Gene Therapy in Human Genetic Diseases

Shao-shuai wu.

1 Key Laboratory of Growth Regulation and Transformation Research of Zhejiang Province, School of Life Sciences, Westlake University, 18 Shilongshan Road, Hangzhou 310024, Zhejiang Province, China.

Qing-Cui Li

Chang-qing yin.

2 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts

3 Program in Molecular Medicine and Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts

Chun-Qing Song

CRISPR/Cas genome editing is a simple, cost effective, and highly specific technique for introducing genetic variations. In mammalian cells, CRISPR/Cas can facilitate non-homologous end joining, homology- directed repair, and single-base exchanges. Cas9/Cas12a nuclease, dCas9 transcriptional regulators, base editors, PRIME editors and RNA editing tools are widely used in basic research. Currently, a variety of CRISPR/Cas-based therapeutics are being investigated in clinical trials. Among many new findings that have advanced the field, we highlight a few recent advances that are relevant to CRISPR/Cas-based gene therapies for monogenic human genetic diseases.

Introduction

The past 20 years have witnessed great progress in genome editing techniques, including meganucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nuclease system. These tools hold great potential for treating human disease, especially genetic diseases beyond the reach of traditional approaches 1 . The CRISPR/Cas system has rapidly become the most popular genome editing platform due to its simplicity and adaptability 2 - 5 .

The CRISPR/Cas system was originally discovered as a prokaryotic adaptive immunity system used to recognize and cleave invading nucleic acids 6 - 8 . Based on this prokaryotic system, scientists have engineered a series of CRISPR/Cas tools for genome editing in mammalian cells, with the list of CRISPR/Cas systems in use continuing to expand. The most commonly used Cas nuclease comes from Streptococcus pyogenes (SpCas9), and belongs to the type II CRISPR system. SpCas9 was the first to be reprogrammed for genome editing in mammalian cells. For specific nucleotide sequence recognition, engineered SpCas9 relies on the guidance of a single-guide RNA (sgRNA). Typically, sgRNA is composed of a scaffold sequence that is bound by the Cas protein, and a custom-designed ∼20 nucleotide spacer that defines the genomic target to be modified. Following hybridization of the spacer to a target genomic sequence that is positioned next to a protospacer adjacent motif (PAM), the target DNA is cleaved, leading to a double-strand break (DSB) 7 - 9 . The Cas-mediated DSB is subsequently repaired by cellular DNA repair machinery via homology- directed repair (HDR) or the non-homologous end joining (NHEJ) pathway. NHEJ can be used to produce insertions and deletions (indels) that disrupt or inactivate the target gene, while HDR can be used for precise nucleotide sequence modifications, such as point mutation correction 10 - 12 ( Figure ​ Figure1 1 a ).

Examples of CRISPR/Cas9 technological advances. (a) Cas9 is directed by single guide RNA (sgRNA) to the target sequence. Double stranded DNA breaks are subsequently repaired by cellular DNA repair machinery via the NHEJ or HDR pathway. (b) dCas9 fused with transcriptional activators or repressors activates or inhibits the expression of a target gene. These systems are called CRISPRa or CRISPRi. dCas9 indicates catalytically inactive dead Cas9, which is able to bind the target DNA without cutting. CRISPRa, CRISPR activators to activate transcriptional process; CRISPRi, CRISPR inhibitors to interference transcriptional process. (c) Base editors are the combination of Cas9 D10A nickase with cytidine or adenine deaminase to induce G->T or A->G transition. Prime editor, different from base editors, is the fusion protein of Cas9 H840A nickase and reverse transcriptase. It can achieve up to 12 types of base-to-base conversions, and targeted insertions and deletions without DSBs or donor DNA templates. pegRNA, prime editing guide RNA.

To date, CRISPR/Cas-based techniques have been applied in various cell types and organisms. For therapeutic genome editing to treat monogenic diseases, CRISPR has the potential to be used directly in patients ( in vivo ) or in human cells ( in vitro ). In this review, we focus on CRISPR strategies used to treat human monogenic diseases, and discuss the challenges associated with these approaches.

Recent advances in CRISPR/Cas technology

Shortly after SpCas9 was applied in mammalian cells, other Cas9 proteins have been studied and developed as genome editing tools. For example, smaller Cas9 proteins derived from Staphylococcus aureus called SaCas9 13 and Neisseria meningitidis called Nme2Cas9 14 exhibit gene editing efficiency comparable to that of SpCas9. These smaller Cas9s are more amenable for in vivo delivery than the large SpCas9 (~4.3 kb).

CRISPR/Cas9 technological advances have also enabled various applications of nuclease-deficient Cas9s, which can bind a specific region of the genome without creating DSBs. For example, catalytically inactive dead Cas9 (dCas9) can be fused with various transcription regulatory domains to create CRISPR activators (CRISPRa) or inhibitors (CRISPRi) that activate or silence the expression of a target gene 15 ( Figure ​ Figure1 1 b ). dCas9 can also be used as a visualization tool. Chen and colleagues have used dCas9 fused to enhanced green fluorescent protein (EGFP) to visualize repetitive DNA sequences using one sgRNA, or nonrepetitive loci using multiple sgRNAs 16 - 18 . In addition, David R. Liu's group has fused D10A Cas9 nickase with either cytidine or adenine deaminase to generate cytidine base editors (CBEs) and adenine base editors (ABEs), respectively. CBEs and ABEs generate transitions between A•T and C•G base pairs without causing high levels of double-stranded DNA cleavage in the target genomic region. Importantly, the Liu's group has extended base editing to utilize H840A Cas9 nickase fused with reverse transcriptase to create prime editors (PEs), which can achieve all possible base-to-base conversions (12 in total), as well as targeted insertions and deletions without DSBs or donor DNA templates 19 ( Figure ​ Figure1 1 c ).

In addition to DNA editing, Feng Zhang's lab has reported that an RNA-targeting CRISPR system based on Cas13 can target and cleave specific strands of RNA, and subsequently developed strategies called REPAIR (RNA Editing for Programmable A to I Replacement) and RESCUE (RNA Editing for Specific C to U Exchange) to edit RNA 20 , 21 . Thus, RNA editing with CRISPR can efficiently modulate target genes at the transcript level in a transient and PAM independent manner. This approach could provide a controllable approach for disease treatment.

Applications of CRISPR in genetic diseases

To date, CRISPR/Cas systems have been used to investigate target genes in genome modification 22 , splicing 23 , transcription 24 and epigenetic regulation 25 , and have been applied in a research setting to investigate and treat genetic diseases 26 , infectious diseases 27 , cancers 28 , and immunological diseases 29 , 30 . Among the exciting advances, translational use of CRISPR/Cas in monogenic human genetic diseases has the potential to provide long-term therapy after a single treatment. In this section, we summarize the recent applications of the CRISPR/Cas system in the generation of disease models and in the treatment of genetic diseases in vitro and in vivo .

Disease modeling using CRISPR/Cas

The generation of disease models is necessary for understanding disease mechanisms and developing new therapeutic strategies. CRISPR/Cas has been widely used for creating disease-related cellular models, such as DMD 31 , aniridia-related keratopathy (ARK) 32 , brittle bone 33 , X-linked adrenoleukodystrophy (X-ALD) 34 , and Alzheimer's disease 35 . Moreover, researchers have created a series of mouse models using CRISPR/Cas that recapitulate DMD 36 , atherosclerosis 37 , obesity and diabetes 38 , RTHα 39 , and Alzheimer's disease 40 ( Table ​ Table1 1 ). One example is the development of a mouse model for ryanodine receptor type I-related myopathies (RYR1 RM), which harbors a patient- relevant point mutation (T4706M) engineered into one allele, and a 16-base pair frameshift deletion engineered into the second allele of the RYR1 gene. Subsequent experiments demonstrated that this mouse model of RYR1 RM is a powerful tool for understanding the pathogenesis of recessive RYR1 RM, and for preclinical testing of therapeutic efficacy 41 . CRISPR/Cas has also been used to generate disease models in large animals, including sheep 42 , rabbit 43 , pig 44 , and monkey 45 . For example, a monkey model was developed to study Parkinson's disease by introducing a PINK1 deletion and revealed a requirement for functional PINK1 in the developing primate brain 45 . CRISPR/Cas technology offers a flexible and user-friendly means of developing disease models to explore the genetic causes of diseases and evaluate therapeutic strategies.

Animal diseases models generated by CRISPR listed in this review.

Disease correction using CRISPR/Cas in model organisms and clinical trials

Monogenic diseases affect a large population of patients. In the ClinVar database, more than 75,000 pathogenic genetic variants have been identified 19 , 46 . Here we summarize recent therapeutic applications of CRISPR/Cas in model organisms and in clinical trials ( Table ​ Table2 2 and Table ​ Table3 3 ).

Preclinical CRISPR Therapy in disease models listed in this review.

CRISPR clinical trials for inherited diseases listed in this review.

Data from https://clinicaltrials.gov/

Hemoglobinopathies

Inherited blood disorders are good candidates for gene therapies because gene therapy can modify the causative gene in autologous hematopoietic stem cells (HSCs) and correct the hematopoietic system. β-thalassemia and sickle cell disease are two genetic blood diseases. β-thalassemia is due to various mutations including small insertions, single point mutations or deletions in β-globin gene, resulting in loss or reduced β-globin synthesis 47 . Sickle cell disease is caused by a Glu->Val mutation in β-globin subunit of hemoglobin 48 , 49 , leading to abnormal hemoglobin S. Re-expressing the paralogous γ-globin genes is a universal strategy to ameliorate both β-globin disorders. The Bauer group applied CRISPR/Cas-based cleavage of the GATA1 binding site of the erythroid enhancer. This approach decreases erythroid expression of the γ-globin repressor BCL11A and in turn increases γ-globin expression. This strategy is therapeutically practicable to produce durable fetal hemoglobin induction 50 - 52 ( Table ​ Table2 2 ).

To date, three clinical trials aiming to treat patients with β-thalassemia and severe sickle cell disease by transfusion of CRIPSR/Cas9 edited CD34+ human HSCs (CTX001) have been initiated by CRISPR Therapeutics in 2018 and Allife Medical Science and Technology Co., Ltd in 2019 ( Table ​ Table3 3 ).

Inherited eye disease

Leber congenital amaurosis (LCA) is a rare genetic eye disease manifesting severe vision loss at birth or infancy. LCA10 caused by mutations in the CEP290 gene is a severe retinal dystrophy. CEP290 gene (~7.5 kb) is too large to be packaged into a single AAV. To overcome this limitation, Editas Medicine developed EDIT-101, a candidate genome editing therapeutic, to correct the CEP290 splicing defect in human cells and in humanized CEP290 mice by subretinal delivery. This approach uses SaCas9 to remove the aberrant splice donor generated by the IVS26 mutation. In the human CEP290 IVS26 knock-in mouse model, over 94% of the treated eyes achieved therapeutic target editing level (10%) when the dose of AAV was not less than 1 × 10 12 vg/ml 53 . Allergan and Editas Medicine have initiated a clinical trial of EDIT-101 for the treatment of LCA10 ( Table ​ Table3 3 ).

Autosomal dominant cone-rod dystrophy (CORD6) is induced by a gain-of-function GUCY2D mutation. CRISPR/Cas components delivered by AAV specifically disrupt the early coding sequence of GUCY2D in the photoreceptors of mice and macaques by NHEJ. This study was the first to successfully perform somatic gene editing in primates using AAV-delivered CRISPR/Cas (up to 13% editing efficiency of GUCY2D mutant gene in macaque photoreceptor), and demonstrated the potential of CRISPR/Cas to cure inherited retinal diseases 54 .

Muscular genetic disease

DMD, caused by mutations in the dystrophin gene, is the most common form of progressive muscular dystrophy, and is characterized by muscle weakness, loss of ambulation, and premature death. Several groups have used NHEJ to bypass a premature stop codon in exon 23 and restore the expression of dystrophin in neonatal and adult mice after local or systemic delivery of CRISPR/Cas components by AAV 55 - 57 . Similarly, CRISPR/Cas- induced NHEJ has been used to treat DMD in a DMD dog model after AAV-mediated systemic delivery of CRISPR gene editing components. 3 to 90% of dystrophin was recovered at 8 weeks after systemic delivery in skeletal muscle, the editing efficiency was dependent on muscle type and the muscle histology was improved in treated dogs 58 . In addition, ABE was delivered locally by intramuscular injection of a trans-splicing AAV to cure DMD in a mouse model 59 . These studies highlight the potential application of gene editing for the correction of DMD in patients.

Congenital muscular dystrophy type 1A (MDC1A), one of neuromuscular disorders, usually appears at birth or infancy. It is mainly featured by hypotonia, myasthenia and amyotrophy. MDC1A is caused by loss-of-function mutations in LAMA2 , which encodes for laminin-α2. To compensate for the loss of laminin-α2, Ronald D. Cohn and his colleagues used CRISPRa to upregulate LAMA1 , which encodes laminin-α1 and is a structurally similar protein to laminin-α2. Upregulation of LAMA1 ameliorates muscle wasting and paralysis in the MDC1A mouse model and provides a novel mutation-independent approach for disease correction 60 .

Genetic liver disease

Hereditary tyrosinemia type I (HTI) patients with loss of function FAH mutations accumulate toxic metabolites that cause liver damage. CRISPR/Cas- mediated HDR has been used to correct FAH mut/mu t in the HTI mouse model by hydrodynamic injection of plasmids encoding CRISPR/Cas components or by combined delivery of AAV carrying HDR template and sgRNA and of nanoparticles with Cas9 mRNA 61 , 62 . VanLith et al. transplanted edited hepatocytes with corrected FAH into recipient FAH-knockout mice and cured HTI mice 63 . Song et al. have used ABE in an adult mouse model of HTI to correct a FAH point mutation 64 . In addition to correcting FAH, several groups have knocked out hydroxyphenylpyruvate dioxygenase (HPD), which acts in the second step of tyrosine catabolism and is an upstream enzyme of FAH, to prevent toxic metabolite accumulation and treat HTI metabolic disease 65 .

Patients with alpha-1 antitrypsin deficiency (AATD) develop liver disease due to a toxic gain-of- function mutant allele, as well as progressive lung disease due to the loss of AAT antiprotease function. CRISPR/Cas-mediated NHEJ has been used to disrupt mutant AAT to reduce the pathologic liver phenotype 66 , while HDR has been used to correct an AAT point mutation 67 .

Congenital genetic lung disease

Congenital genetic lung diseases include cystic fibrosis and inherited surfactant protein (SP) syndromes 68 . Monogenic lung diseases caused by mutations in SP genes of the pulmonary epithelium show perinatal lethal respiratory failure death or chronic diffuse lung disease with few therapeutic options. Using a CRISPR fluorescent reporter system, scientists precisely timed intra-amniotic delivery of CRISPR/Cas9 components into a prenatal mouse model with the human SP gene SFTPC I73T mutation to inactivate mutant SFTPC I73T gene through NHEJ. Prenatal gene editing in SFTPC I73T mutant mice rescued lung pathophysiology, improved lung development, and increased survival rate to 22.8%. For intra- amniotic delivery, the amniotic cavity of embryonic day 16 mouse fetus, in which fetal breathing movements are optimal for fetal lung editing, was injected. After prenatal CRISPR delivery, embryonic day 19 fetus achieved up to 32% SFTPC wild-type airway and alveolar epithelial cells in SFTPC I73T mice, rescued lung pathophysiology by immunohistology, improved lung development by reducing the synthesis of mis trafficked SFTPC mutant proprotein, and increased survival rate to 22.8% 69 .

Cystic fibrosis is another life-threatening monogenic lung disease caused by mutations in CFTR gene 70 . Researchers applied CRISPR to precisely corrected CFTR carrying homozygous F508 deletion (F508del) in exon 10 in the induced pluripotent stem cells (iPSC) separated from cystic fibrosis patients 71 and the overall correction efficiency is up to 90% using piggyBac transposase as selection marker. Xu group applied the electroporation of CRISPR/Cas RNP and achieved more than 20% correction rate in patient-derived iPSC cell line with F508del mutation 72 . As expected, CRISPR-induced genetic correction leads to the recovery of CFTR function in airway epithelial cells or proximal lung organoids derived from iPSC.

Genetic deafness

At least half of all cases of profound congenital deafness are caused by genetic mutations and genetically inherited. Approximately 120 deafness- associated genes have been identified, but few treatments are available to slow or reverse genetic deafness 73 . Recently, David R. Liu's group employed cationic lipid-mediated in vivo delivery of Cas9-guide RNA complexes to disrupt the dominant deafness-associated allele in the humanized transmembrane channel-like 1 (Tmc1) Beethoven (Bth) mouse model and ameliorated the hearing loss in these animals 74 . David P. Corey's group screened 14 Cas9/sgRNA combinations and identified that SaCas9-KKH/gRNA could specially and safely recognize mutant Tmc1 but not wildtype allele in vitro and in vivo , which provides a strategy to efficiently and selectively disrupt the dominant single nucleotide mutation rather than the wild-type alleles 75 .

Overcoming limitations of CRISPR/Cas-based gene therapy

Extensive work is being done with CRISPR/Cas in disease research and recent reviews had summarized the advantages of CRISPR/Cas 76 , 77 . The safety and efficacy of CRISPR/Cas9-based gene therapies need to be evaluated and refined before these therapies are applied in patients 78 . One of the common limitations for CRISPR/Cas is that not all the mutation locus harbors the PAM motif, which the target recognition relies on. Besides, the challenges for using CRISPR/Cas as gene therapy include editing at off-target genomic sites, delivery vehicle, immunogenicity, and DNA damage response.

Off-target effects of CRISPR/Cas

Despite significant advances in understanding the CRISPR/Cas9 system, concerns remain regarding off-target effects. Indeed, several groups found a tradeoff between activity and specificity of CRISPR/ Cas9, identifying off-target DNA cleavage by genome wide deep sequencing technique 79 - 81 . Moreover, CBEs and ABEs cause transcriptome-wide off-target RNA editing 82 , 83 . Thus, unwanted off targets are concerns for the application of CRISPR. However, off-target effects can be reduced with sgRNA selection and optimization. Also, verification of in vivo off-targets (VIVO) can be used for defining and quantifying off-target editing of nucleases in whole organisms 84 . The recently developed anti-CRISPR proteins could conditionally control the activity of the CRISPR system 85 - 88 , which may show the potential in reducing off-target effects. The development of more sensitive methods is necessary for detecting off-target editing at both genome and transcriptome levels.

In vivo delivery of CRISPR/Cas

AAV is the most widely used in vivo delivery of CRISPR/Cas. However, AAV has a limited packaging capacity, hindering all-in-one delivery of CRISPR/ Cas components, in particular larger Cas-derived base editor and prime editor. This has led to continued development of smaller Cas9 orthologues like SaCas9 13 . For instance, saCas9 or NmeCas9 and sgRNA have been combined into a single AAV vector for inducing indels to correct disease. For disease correction by HDR or base editors, dual AAV or split AAV vectors can be used to circumvent packaging size limitations 89 , 90 . A disadvantage of such an approach is the requirement of uptake and expression of both AAV vectors into the same cell at roughly the same time to ensure intracellular Cas9:sgRNA complex formation.

CRISPR/Cas components can also be delivered by non-viral methods, for instance, Cas9 mRNA and sgRNA can be delivered to mouse liver by nanoparticles 62 . But the external and internal barriers for nanoparticles entering the cell and nucleus must be considered. Currently, nanoparticles carrying CRISPR/Cas components are largely applied to mice and delivered into liver. Because the liver contains fenestrated capillary endothelia. Further improvement of nanoparticle-based CRISPR/Cas components delivery systems is needed for other target tissues.

Immune response stimulated by CRISPR/Cas

The application of CRISPR/Cas systems raises concerns over immunogenicity of the bacterially- derived Cas9 protein 91 . In a recent study, Charlesworth et al. demonstrated that anti-Cas9 responses are present in healthy human adults 92 . In 34 human blood samples, anti-Cas9 IgG antibodies were detected against SaCas9 (79% of samples), and against SpCas9 (65% of samples). The immunogenicity of SpCas9 in healthy humans has been reported by Michael's group. Specifically, they found that high prevalence of effector T cells towards SpCas9 exist prior to the delivery of SpCas9 93 . This issue will need to be addressed in the clinical applications of CRISPR/Cas.

DNA damage response activated by CRISPR/Cas

In CRISPR/Cas gene editing via NHEJ and HDR, DSBs are generated at the target sites. DBS- based repair activates a p53-dependent DNA damage response and induces transient cell cycle arrest, leading to a decrease in efficiency of template- mediated precision genome editing 94 . In human pluripotent stem cells, p53-deficient cells are more susceptible to CRISPR-mediated modification 95 . These findings suggest that, during clinical trials, CRISPR-engineered cells or organs in patients should be monitored for p53 function. To avoid DSB triggered p53-mediated response, base editors (ABE and CBE) and prime editors can be applied for precision gene editing-mediated target gene correction.

Conclusion and perspectives of using CRISPR/Cas in the clinic

CRISPR/Cas has already shown great potential in generating disease models and correcting monogenic disease mutations. The CRISPR disease models can accelerate the discovery and development of drug targets. In addition to the widely used type II CRISPR/Cas systems, continued discovery and development of CRISPR systems from prokaryotic species has generated new technologies. For example, DN1S-SpCas9 fusion protein blocks local NHEJ events and increases HDR frequency 96 . Moreover, Cas13a-based RNA-targeting tools enable RNA changes that are temporally and spatially controllable, and will broaden and facilitate the application of RNA therapy in human diseases. Before the application of CRISPR for human disease correction, efforts are needed to optimize and maximize the editing efficiency as well as minimize off-targets and develop novel tools to specifically deliver the CRISPR components to the target tissue for gene editing 97 , 98 . As CRISPR/Cas-based gene therapy enters clinical trials ( Table ​ Table3 3 ), this technology holds great potential for treating genetic diseases particularly for the present incurable ones and enhancing cell therapies.

Acknowledgments

The authors thank Craig. Mello, Scot Wolfe, En-Zhi Shen, and Erik. Sontheimer for discussions, Suet-Yan Kwan and Emily Haberlin for editing the manuscript, and Ya-Ping Shen for raw figure preparation. Wen Xue was supported by grants from the National Institutes of Health (DP2HL137167, P01HL131471 and UG3HL147367), American Cancer Society (129056-RSG-16-093), the Lung Cancer Research Foundation, and the Cystic Fibrosis Foundation. Chun-Qing Song was supported by start funding of Westlake University.

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• Review Article
• Published: 05 October 2020

Modulating gene regulation to treat genetic disorders

• Navneet Matharu   ORCID: orcid.org/0000-0002-9426-1484 1 , 2 , 3 &
• Nadav Ahituv   ORCID: orcid.org/0000-0002-7434-8144 1 , 2  

Nature Reviews Drug Discovery volume  19 ,  pages 757–775 ( 2020 ) Cite this article

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Over a thousand diseases are caused by mutations that alter gene expression levels. The potential of nuclease-deficient zinc fingers, TALEs or CRISPR fusion systems to treat these diseases by modulating gene expression has recently emerged. These systems can be applied to modify the activity of gene-regulatory elements — promoters, enhancers, silencers and insulators, subsequently changing their target gene expression levels to achieve therapeutic benefits — an approach termed cis -regulation therapy (CRT). Here, we review emerging CRT technologies and assess their therapeutic potential for treating a wide range of diseases caused by abnormal gene dosage. The challenges facing the translation of CRT into the clinic are discussed.

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Acknowledgements

This article was supported in part by grants 1R01DK090382 and 1R01DK124769 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the Simons Foundation Autism Research Initiative grants 629287 and 564256, the University of California, San Francisco (UCSF) School of Pharmacy 2017 Mary Anne Koda-Kimble Seed Award for Innovation and the Innovative Genomics Institute RIDER award 2019. The authors regret they could not include and highlight the comprehensive list of citations of their fellow scientists due to space limitations.

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N.A. is an equity holder of, and a scientific advisor for Encoded Therapeutics, a gene regulation therapeutics company. N.A. and N.M. are cofounders of Enhancer Therapeutics Inc. and co-inventors on a related patent (Publication number WO/2018/148256). N.M. and N.A. are co-inventors on a patent (US Patent US2018017186) submitted by the University of California, San Francisco, that covers gene therapy for haploinsufficiency.

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(CREs). DNA sequences that regulate the transcription of a neighbouring gene.

Assembly of the nucleic acids and capsid during virus generation.

Irreversible and unintended DNA changes caused mainly due to off-targeting by DNA targeting modules with functional nucleases.

Physical DNA–DNA interaction in the genome within 3D nuclear space.

Particles that are between 1 and 100 nm in diameter.

A route of delivery via injection into the cerebrospinal fluid in cerebral ventricles.

A specific 3-bp DNA sequence that has more copies than normal in the genome.

The proportion of the therapeutic agent upon administration that has an active effect.

The effects arising due to non-specific and unintended targeting of DNA targeting modules such as zinc fingers, transcription activator-like effector (TALE) and CRISPR in the genome.

The methods of administration of a therapeutic agent based on the site of action.

(Also known as a viral envelope). The proteinaceous shell that packages the genetic material of the virus. Its structure is important in determining viral stability, delivery and host interactions.

The adaptive immune response of the body due to pre-exposure to an antigen.

(Adeno-associated virus serotypes). The variations in the capsid surface proteins of an adeno-associated virus that can define its transduction efficiency in different tissue or cell types.

The blood–brain barrier is the membrane made from endothelial cells surrounding the blood vessels that selectively allows solutes to transfer from the blood to the central nervous system.

A route of delivery via injection into the spinal canal in order to avoid the blood–brain barrier selective permeability.

A route of delivery into the vitreous humour of the eye.

Circular DNA that is not integrated in the genome.

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Matharu, N., Ahituv, N. Modulating gene regulation to treat genetic disorders. Nat Rev Drug Discov 19 , 757–775 (2020). https://doi.org/10.1038/s41573-020-0083-7

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Key clues to DNA repair mechanism might lead to new cancer treatments

by Tokyo Metropolitan University

Researchers from Tokyo Metropolitan University have identified key factors in the mechanism behind DNA repair in our bodies. For the first time, they showed that the "proofreading" portion of the DNA replicating enzyme polymerase epsilon ensured safe termination of replication at damaged portions of the DNA strand, ultimately saving DNA from severe damage. This new knowledge arms scientists with ways to make anti-cancer drugs more effective and may provide new diagnostic methods.

Our DNA is under attack. Every day, around 55,000 single-strand breaks (SSBs) appear in the strands making up DNA helices in individual cells . When polymerases, molecules that replicate DNA strands, try to make new helices from strands with breaks in them, they can break the helix, creating what's known as a single-ended double-stranded break (seDSB).

Thankfully, cells have their own ways of dealing with strand damage. One is homology directed repair (HDR), where double stranded breaks are fixed. Another is "fork reversal," where the replication process is reversed, preventing the single-strand nicks turning into DSBs in the first place.

The exact mechanism behind fork reversal remains unknown. Understanding how DNA damage is prevented is paramount not only to prevent cancers, but also ensure the effectiveness of cancer drugs which rely on DNA damage. Take camptothecin (CPT), an anti-cancer drug that introduces lots of single-strand breaks; since cancer cells tend to replicate quicker, they create lots of seDSBs and die out, leaving normal cells less harmed.

Now, an international team led by Professor Kouji Hirata of Tokyo Metropolitan University have shed new light on how fork reversal works. They focused on polymerase epsilon, an enzyme responsible for making new DNA from a portion of the DNA which has unzipped. They discovered that the exonuclease, the "proofreading" portion of the polymerase that ensures copy accuracy, played a key role, a new, rare insight into the largely unknown molecular mechanism behind fork reversal.

The paper is published in the journal Nucleic Acids Research .

First, the team found that cells that are deficient in the exonuclease part showed strong susceptibility to exposure to CPT. Suppression of a factor known as PARP, the only other player known to affect fork reversal, also led to increased cell death. However, when both were suppressed, there was no further increase in cell death beyond what was seen with PARP. This suggests that PARP and the polymerase epsilon exonuclease work together to trigger fork reversal.

In addition, the team studied cells with the gene coding for BRCA1 (the breast cancer susceptibility protein) disrupted; additional deficiency of the exonuclease caused drastically increased sensitivity to CPT, far more than expected from either defect. Since BRCA1 deficiency is linked to a high risk of breast cancer, the exonuclease might be targeted to make drug treatments more effective.

The researchers have shown that drugs targeting the polymerase epsilon exonuclease can amplify the effect of anti-cancer drugs . Equally importantly, defects to the exonuclease have also already been seen in a wide range of cancers, including intestinal cancer; this makes it likely that such cells have impaired fork reversal capability, a promising target for future diagnostics as well as treatments.

Journal information: Nucleic Acids Research

Provided by Tokyo Metropolitan University

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How Gene Therapy and Research Is Helping Us Treat Brain Disease

Learn how gene therapy is helping researchers build a foundation for better, more effective brain disease treatments.

Research breakthroughs in recent decades have shown that gene therapy has the potential to be an effective treatment and diagnostic tool for various brain diseases. While there are still clear limitations and challenges to gene therapy for brain disease, genetic research has enabled us to develop deeper understandings of disease formation and progression and risk factors for multiple diseases and disorders. Below we discuss how some of these discoveries will help us develop better treatments for brain disease in the future.

How does gene therapy for brain disease work?

While brain diseases have many different causes, including one’s environment and complex biological factors, some are caused by a genetic mutation. When a gene is abnormal in some way, it affects a person’s normal bodily functions and development. By identifying specific gene mutations through genetic testing, researchers have been able to pinpoint the mutations responsible for many brain diseases.

Gene therapy works to correct a genetic mutation. A delivery vehicle, typically a harmless virus called a viral vector, provides the normal version of the mutated gene , which then over time distributes throughout the brain. While researchers have had some success using gene therapy to treat neurodegenerative diseases in rodents, it’s much more complex to apply the same treatment to humans. One challenge is the blood-brain barrier . This barrier is a protective mechanism around the brain. To deliver the normal version of a gene, a viral vector that is injected in the blood has to be able to cross the blood-brain barrier and reach different parts of the brain.

While it’s not quite the same as gene therapy, identifying the specific genes responsible for certain brain diseases has also laid the groundwork for advanced diagnosis and treatment options. When we know which genes correspond to specific diseases, doctors can make more accurate diagnoses and use more effective, targeted treatments. Gene testing can be especially helpful when diagnosing rare genetic brain diseases with presenting symptoms that may closely overlap with other more common disorders.

Can gene therapy cure neurological disorders?

Currently, no—but researchers are trying to develop more effective treatments that target DNA and specific gene mutations. Genetic research and tools like genetic testing may help in the development of new treatments for brain disease, even if those treatments aren’t yet complete cures. It is a victory when gene therapy leads to a significant improvement in brain disease symptoms or a clearer understanding of the mechanisms through which a disease impacts brain function. 

Breakthroughs in gene therapy help researchers learn more about how different delivery vehicles work, how to adjust treatments to make them safer and more affordable, and how to apply these findings to other diseases. Let’s take a closer look at some of the ways genetic research is making progress in brain disease diagnosis and treatment.

Genetic Research Leading to the Development of New Medications

Genetic research can lead to a greater understanding of why a disease develops and how it progresses—and in turn, how to more effectively treat it. Since 1993, scientists have known the genetic cause of Huntington’s disease —a hereditary (inherited) neurodegenerative brain disease that interferes with thinking, behavior, and movement—but there have been no treatments developed specifically targeting this genetic basis for the disease. 

Now, gene therapy is offering hope that new drug treatments could slow or stop the effects of mutated genes in diseases like Huntington’s. The development of drugs called antisense oligonucleotides (ASOs) for Huntington’s disease is one example of how research findings can lead to new drug therapies. 

ASO drugs are pieces of DNA or RNA that link to the disease-causing proteins produced by mutated genes. They can then stop accumulation of these harmful proteins or can replace missing ones in order to rebalance protein levels. While ASO drugs are invasive and not yet widely used, they represent an important step toward improved treatments for Huntington’s disease—and perhaps other brain diseases in the future.

Gene Therapy Shows Promise for Treating Certain Neurodegenerative Diseases

A recent clinical trial shows promise for delivering gene therapy directly to specific parts of the brain by viewing its effects in real time using magnetic resonance imaging (MRI). For this trial, scientists at Ohio State University administered gene therapy to children with a rare neurodegenerative brain disease. 

The disease involves a deficiency that affects the body’s ability to make dopamine and serotonin, which are neurotransmitters or “messengers” in the brain. When this happens, it can cause developmental delays, behavioral problems, and movement issues.

After receiving gene therapy directly to the midbrain, children in the clinical trial saw improvement in symptoms like eye spasms, sleep disturbances, and issues with head control. With the success of this trial, researchers are hopeful that brain-delivered gene therapy could be used to treat other more common neurodegenerative diseases like Alzheimer’s and Parkinson’s disease.

Genetic Testing May Help Diagnose Brain Disease Earlier in Life

The connection between gene mutations and specific brain diseases can help with earlier diagnosis and treatment. Genetic testing , often done through a blood sample, can detect any abnormal or mutated genes. This information can help doctors identify whether a person has a specific disease, is at risk to develop a disease, or is a carrier of a gene mutation they could pass along to their children.

Gene mutations can either cause a condition or increase a person’s risk of developing it, and often multiple genes can play a role in a specific brain disease. There are also different types of genetic testing: diagnostic, when someone has symptoms of a disorder, and presymptomatic, when a person doesn’t have symptoms but has an increased risk based on family history or environmental factors.

As researchers learn more about major and minor gene mutations and which ones may cause certain disorders, genetic testing could become a more helpful tool. Because genes are passed down through family members, the findings of a genetic test can have an impact beyond the person who undergoes testing. It’s also crucial to consider how the results of genetic testing may impact a person’s life and inform potential treatment options. While testing can provide answers, it can also open up more questions.

These three areas—development of new medications, new treatment methods, and genetic testing—are evidence that genetic research has the potential to revolutionize the the way we understand, diagnose, and treat many different brain diseases. This type of research helps make progress toward more effective treatments for all brain diseases. Because the brain is interconnected, as we discover more about genetic mutations and gene therapy for one disease, we will be able to apply these learnings to help treat other brain diseases.

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“DNA breaks and repair events occur thousands of times per day in each of us,” said Catherine Freudenreich. Image: CC by SA/Wikipedia

How DNA Repair Can Go Wrong and Lead to Disease

Researchers uncover how the success of natural DNA repair depends on surrounding sequences

We often come to an understanding of what causes a disease. We know, for example, that cancers are caused by mutations at critical locations in the genome, resulting in loss of control of cell growth. We know that the onset of Huntington’s disease, and other diseases that lead to muscle wasting and loss of coordination and balance, are linked to the expansion of short, repeated DNA sequences.

What we don’t know is how these genetic events come about. In a study published in the journal Nature Communications , Catherine Freudenreich, professor and chair of the Department of Biology at Tufts, and her team of researchers uncovered mechanisms by which the natural process of DNA repair fails and mutations arise, opening up potential paths for understanding the origins of many diseases and the development of therapies to treat them.

“DNA breaks and repair events occur thousands of times per day in each of us,” said Freudenreich. “Most of the time, repair works the way it should. But we now have a better understanding of how things can go wrong, and we can apply this knowledge to new therapeutic strategies.”

Erica Polleys, a postdoctoral researcher in the Freudenreich Lab , focused on the gene repeats found in Huntington’s disease—a long repetition of 60 or more copies of the sequence cytosine-adenine-guanine, or CAG, on one strand of the DNA and cytosine-thymine-guanine, or CTG, on the complementary strand.

“We’ve engineered yeast to have a CAG repeat sequence, because their cells are similar to human cells in many ways and they are easy to work with,” said Polleys, “We can use simple model organisms to uncover how cells deal with repairing breaks and errors near repetitive sequences and use that knowledge to get an understanding of how human diseases like Huntington’s disease or cancers develop.”

Source: Freudenreich Lab

It has been widely understood that DNA repeats can form structures that branch away from the linear chain of DNA, much like a cord or a hose will form branching twists if wound too tightly. These branches form barriers to natural replication and repair and introduce errors in the genome.

Freudenreich and Polley’s examination of the mechanisms of DNA repair near Huntington’s disease-like repeats revealed that repeats would expand or contract in number depending on which strand of the double stranded DNA was being repaired.

When the CAG side of the strand was being removed during a repair, the repeats contracted, and the DNA was at risk of large deletions, often fatal to the cell. When its complementary CTG strand was being removed and repaired, the repeats expanded, but only by one or two copies at a time. The researchers hypothesize that this is how the repeats accumulate in neurons of Huntington’s disease patients.

A key observation was that DNA breaks near expanded repeats occurred a lot more frequently when the repeats were exposed as a single strand of DNA, something that occurs during many repair processes. This “fragile site” can lead to deletion of a nearby important gene, which can kill the cell, or result in diseases like cancer.

DNA fragility can happen near other sequences that make the DNA form branched twists. Genomic analysis could reveal more mutation hotspots that lead to cancer, or the accumulation of repair errors that lead to many conditions of aging.

Observing changes in repeat length provided more detail about what was happening, but not how it was happening. The Tufts team picked apart the repair mechanism further, deleting some proteins to see how they affected the fragility of the yeast genome.

Removal of one protein led to a faster recruitment of other proteins that untwist the DNA, leading to more successful repairs. A second protein was found that may help stabilize the DNA as it is being repaired, like fitting a sleeve over a hose to prevent it from twisting up.

The researchers point to the human equivalent of these yeast proteins, with the possibility that their inhibition or enhancement could lead to safer DNA repair and replication in patients with cancer or DNA repeat diseases, like Huntington’s, myotonic dystrophy, and Friedrich’s ataxia.

Study Shows Where Damaged DNA Goes for Repair

Mapping the Kinks in Faulty DNA

Uncovering the Secrets of Resilience in Nature

Identification of disease-causing proteins leads to new potential treatments for diseases like diabetes

New research has identified hundreds of proteins that might contribute to the onset of common, chronic metabolic diseases such as type 2 diabetes, and consequently pathways to potential treatments.

The study, published in Nature Metabolism , was led by an international research team from the Medical Research Council (MRC) Epidemiology Unit, University of Cambridge, the Precision Healthcare University Research Institute (PHURI) at Queen Mary University of London, and the Berlin Institute of Health at Charité (BIH) at Universitätsmedizin Berlin in Germany.

The researchers successfully linked more than 900 regions in the human genome to almost 3000 proteins in our blood, with many of these not previously identified. The team then applied these findings to existing genetic studies for hundreds of diseases and found more than 500 gene-protein-disease links.

For example, the team showed for the first time that people with high levels of a hormone called GRP are less likely to develop type 2 diabetes, most likely because it decreases the chances of becoming overweight. This 'proteogenomic' evidence supports GRP as a potential target for the prevention and/or treatment of diabetes.

The study has led to a greater scientific understanding of hundreds of genome regions, which open the door to more targeted and ultimately successful treatment options in the future as proteins are essential functional units of the human body and the most common target of drugs that exist today.

Senior author Professor Claudia Langenberg, director of the Precision Healthcare University Research Institute (PHURI) at Queen Mary University of London and MRC Investigator and program lead at the MRC Epidemiology Unit at the time of the study, said: "Thousands of regions in our genome have been identified to increase our risk for developing different diseases, but for most of them we have a poor understanding of why this is.

"By measuring and integrating information on thousands of proteins in human plasma, we were able to create robust links between the genes that encode these proteins to many different diseases and demystify about 200 regions. This really narrows down the potential therapeutic targets at each genomic region, often a bottleneck for translation of genomic discoveries."

Senior author Professor Maik Pietzner, Professor at the PHURI and group leader at the Berlin Institute of Health at Charité (BIH), said "In an additional promising example, we identified a protein, called DKKL1, to be involved in multiple sclerosis that reinforces the depletion of certain immune cells -- B-cells -- as intervention.

"These early-stage results are exciting and show the potential of such technologies for drug discovery, not just for metabolic diseases."

Mine Koprulu lead author of the study, Gates scholar and PhD student at the MRC Epidemiology Unit, said: "The biological mechanisms underlying diseases are not always very well understood. To address this issue, we systematically linked genetic variation, blood protein levels and disease risks in this study to be able to differentiate proteins which are likely to cause a disease, for example diabetes, from those which may only be a result of diseases. Identifying causal proteins is important because only interventions on causal proteins will lead to safe and effective treatments. We are hugely grateful to the EPIC Norfolk volunteers and team who made this research possible."

• Human Biology
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• Chronic Illness
• Immune System
• Alzheimer's Research
• Diabetes mellitus type 2
• Personalized medicine
• Diabetes mellitus type 1
• Stem cell treatments
• Adult stem cell

Story Source:

Materials provided by Queen Mary University of London . Note: Content may be edited for style and length.

Journal Reference :

• Mine Koprulu, Julia Carrasco-Zanini, Eleanor Wheeler, Sam Lockhart, Nicola D. Kerrison, Nicholas J. Wareham, Maik Pietzner, Claudia Langenberg. Proteogenomic links to human metabolic diseases . Nature Metabolism , 2023; DOI: 10.1038/s42255-023-00753-7

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