GENE EDITING TECHNOLOGY - Harnessing a Cell’s Natural DNA Repair Process to Develop Medicines With Higher Levels of Precision & Durability

INTRODUCTION

The ability to target and modify the human genome, or alter its functionality, has transformed molecular biology-based re­search and opened up the possibility of treating a wide range of genetic diseases. Recent progress in the development of gene ed­iting technologies, including those based on zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most notably CRISPR-Cas9-associated nucleases, has taken gene editing from concept and discovery stage to clinical use in human trials.

While gene editing technologies have significant potential, many of the current gene therapies offer limited benefit due to lack of durability and the inability to target pediatric indications. But a new nuclease-free approach to gene editing is emerging and advancing through clinical development, raising the prospect of revolutionizing the treatment of many rare and devastating dis­eases that represent significant areas of unmet need in global health. Researchers at LogicBio Therapeutics are working to develop next-generation gene delivery and genome editing tech­nologies with the goal of addressing limitations and supporting treatment of diseases that affect patients at any age, from infancy throughout adulthood.

In many rare genetic diseases, such as inborn errors of me­tabolism, symptoms present in the first year of life and progress rapidly, often leading to significant and potentially irreversible consequences and even death. Early intervention is critical.

A major challenge in applying traditional gene therapy ap­proaches in the pediatric population is the proliferation of cells in the growing tissues of a child, which dilutes the therapeutic benefit. With existing gene therapy technologies, the corrective genes do not integrate into a patient’s chromosomes but remain floating inside the nucleus. This means that the corrective gene is not carried through to successive generations when cells divide and, as a result, the therapeutic effect is diluted over time, espe­cially in children, or in cases when the tissue is regenerating, such as in a damaged liver.

Gene editing technologies including CRISPR-Cas9, TALENs, and zinc fingers use engineered nucleases to cut a patient’s DNA and remove or insert a corrective gene. But this process is often not precise and has been shown to lead to off-target effects and uncontrolled deletions or insertions in a patient’s chromosomes. These unintended changes to a patient’s DNA can increase the risk of genomic instability, cancer, and other genotoxicities. The use of nucleases, which are typically derived from bacteria, can also raise the risk of provoking a strong immune response in pa­tients.¹

THE POWER OF GENERIDE^TM

LogicBio’s proprietary GeneRide^TM technology is a new genome editing approach that harnesses a cell’s natural DNA re­pair process, known as homologous recombination, to insert a corrective copy of the gene (or transgene) at a precise spot in a patient’s genome. If successful, the gene then persists as an integral part of a patient’s DNA as cells divide. GeneRide is nuclease-free, site-specific genome editing technology with the poten­tial to enable durable expression and lower risk of harmful off target integration. The GeneRide technology was born out of the Kay Lab at Stanford University by LogicBio’s co-founders Mark Kay, MD, PhD, Adi Barzel, PhD, and Leszek Lisowski, PhD, MBA, whose goal was to create an approach for precise and durable genome editing that could provide safety and effi­cacy benefits.

GeneRide is designed to work in a six-step process:

1. A synthetic, non-pathogenic adeno-as­sociated viral (AAV) vector is used to deliver the corrective transgene to the nuclei of a patient’s cells via an infu­sion.
2. Two homology guides – strands of DNA several hundred base pairs long that are designed to precisely match a specific stretch of a patient’s genome – flank the corrective transgene.
3. Upon sensing the therapeutic DNA in the nucleus, the cell’s natural DNA re­pair machinery responds and inte­grates the corrective transgene at a specific site in the patient’s genome. The transgene is inserted in the same place every time – in the chromosome and at the gene that corresponds to the DNA sequence encoded in the homol­ogy guides. For LogicBio’s liver-tar­geted therapies, this specific location for integration is within the albumin locus. Albumin is the most abundant protein in circulation and the most highly expressed gene in the liver. This high expression level is driven by the endogenous albumin promoter, which is very robust and tissue specific.
4. When the therapeutic transgene is in­tegrated into the albumin locus, down­stream of the albumin coding region, it can hitch a ride on the endogenous albumin promoter to drive expression of the corrective transgene a patient has been lacking, without disrupting al­bumin production.
5. By using a 2A peptide – which is a short chain of amino acids that induces ribosomal skipping during translation of a protein in a cell – the cell produces albumin and the transgene as two sep­arate proteins and modifies albumin with a small “tag.” This tag enables LogicBio to monitor GeneRide activity in a patient’s body in a non-invasive way. The tagged albumin protein can be easily detected in the circulation as a surrogate for site-specific integration and protein expression.²
6. Shortly after treatment, the modified cells begin producing the therapeutic protein to combat the target disease.

The GeneRide platform is being lever­aged to develop therapies that target dis­eases that cannot be treated by current genetic medicines, including rare genetic diseases such as methylmalonic acidemia (MMA) and Crigler-Najjar syndrome. To target different genetic diseases, the goal is for a different corrective gene to be sub­stituted within the GeneRide construct while largely maintaining all other compo­nents of the system.

LB-001: IN VIVO GENE INSERTION FOR EARLY INTERVENTION IN MMA

LogicBio chose to first focus on MMA, a rare autosomal recessive disease, because of the potential benefits that GeneRide can provide by treating patients very early in the course of the disease. MMA affects approximately one in 50,000 newborns in the US.³ It can be caused by mutations in several genes, but a mutation in the mitochondrial enzyme methyl­malonyl-CoA mutase (MMUT) gene is the most common.⁴ Mutations in this gene, which provides instructions for making the MMUT enzyme, prevent the body from properly processing certain fats and pro­teins and can lead to the toxic buildup of methylmalonic acid and other disease-causing metabolites. Patients with severe MMA may present with symptoms at birth including poor feeding, vomiting, hypoto­nia, respiratory distress and progressive encephalopathy.^3-5 They are also at in­creased risk of neurological symptoms, failure to thrive, intellectual disability, se­vere infections, and progressive renal in­sufficiency.

To manage symptoms, patients must adopt a severely restrictive, low-protein, high-calorie diet, often through a feeding tube.^4,5 Even with aggressive manage­ment, these patients often experience life-threatening metabolic crises that can cause permanent neurocognitive damage. In some cases, patients may need to un­dergo liver transplantation, an invasive and high-risk procedure that is not widely available and that presents lifelong health ramifications. Due to the need for early in­tervention, newborns are screened for MMA in every state in the US.^5,6

Although newborns are screened for MMA and diagnosed early in life, unfortu­nately, there are currently no approved treatments that can target the root cause of the disease. Patients can only try to manage symptoms, leaving them and their families with an enormous burden and no medications to help.

LB-001 is an investigational, first-in-class, single-administration, in vivo genome editing therapy in development for early intervention in MMA, leveraging the GeneRide platform. LB-001 is de­signed to non-disruptively insert a correc­tive copy of the MMUT gene into the albumin locus to drive lifelong therapeutic levels of MMUT expression in the liver, the main site of MMUT expression and activity. The investigational therapy is delivered to hepatocytes via a liver-targeted, engi­neered recombinant AAV vector (rAAV-LK03).

In preclinical studies, LB-001 was shown to be safe and demonstrated trans­duction of hepatocytes, site-specific ge­nomic integration, and transgene expression. LB-001-corrected hepatocytes in a mouse model of MMA demonstrated preferential survival and expansion (selec­tive advantage), thus contributing to a pro­gressive increase in hepatic MMUTexpression over time. In MMA mice, treat­ment with LB-001 resulted in improved growth, metabolic stability and survival.^7,8

LB-001 is now being evaluated in MMA patients in a Phase 1/2 clinical trial, called the SUNRISE trial, an open-label, multi-center study designed to assess the safety and tolerability of a single intra­venous infusion of LB-001 in pediatric pa­tients with MMA characterized by MMUT mutations. With the aim of evaluating LB-001 at an early age, before irreversible damage has occurred, the SUNRISE trial is designed to enroll up to eight patients with ages ranging from six months to 12 years and evaluate a single administration of LB-001 at two dose levels (5 x 1013 vg/kg and 1 x 1014 vg/kg). This is believed to be the first in vivo genome editing therapy de­livered systematically to pediatric patients and represents a key step in the effort to treat children suffering from early onset genetic diseases such as MMA.

BROAD POTENTIAL OF GENERIDE IN OTHER GENETIC DISEASES

The GeneRide technology platform is modular in nature, meaning it has the po­tential to be used to develop multiple genome editing therapies generally using similar principal components. At the Euro­pean Society of Gene & Cell Therapy, or ESGCT, conference in October 2021, LogicBio presented preclinical data that validates previous research in MMA and highlights selective advantage in two ad­ditional indications that are also charac­terized by intrinsic liver damage: hereditary tyrosinemia type 1 (HT1) and Wilson disease.

Selective advantage enables healthy, edited hepatocytes carrying a corrective gene to survive and reproduce better than the endogenous mutated hepatocytes and to ultimately repopulate a part or whole of a diseased liver. In all three of the disease mouse models, expansion of the corrected healthy hepatocytes correlated with im­proved diseased markers.

In the HT1 models with acute liver damage, the data showed that GeneRide-corrected hepatocytes repopulated the en­tire liver within four weeks post-administration, replacing the dis­eased hepatocytes with corrected hepato­cytes. HT1 mice are deficient in the gene encoding fumarylacetoacetate hydrolase (FAH), which is required to metabolize the amino acid tyrosine, resulting in the accu­mulation of toxic metabolites. HT1 mice that received the GeneRide-FAH vector were no longer reliant on the current stan­dard of care for the disease, and demon­strated restored normal body growth, liver function, and undetectable succinylace­tone levels, one of the toxic metabolites that accumulates in patients with HT1. Compared to the current standard of care, treatment with the GeneRide vector re­sulted in superior succinylacetone reduc­tion and lower alfa-fetoprotein levels, a clinically validated biomarker for hepato­cellular carcinoma and another risk factor for untreated HT1 patients.

Wilson disease results from a defect in copper transport, leading to toxic accu­mulation of copper and damage to tis­sues. In a Wilson disease mouse model, GeneRide-corrected hepatocytes repopu­lated the liver over time, and treated mice showed improvements in liver function, hepatomegaly, and urinary copper excre­tion.

Selective advantage and expansion of corrected hepatocytes was observed in these preclinical models, demonstrated by detection of increasing levels of a tagged albumin protein, albumin-2A, a technol­ogy-related biomarker indicating site-spe­cific gene insertion and protein expression, as well as immunohistochemistry for the corrective protein in liver sections. Results presented at ESGCT also showed increas­ing levels of albumin-2A correlated with increased expression of the corrective gene and improved disease burden. LogicBio believes that these data support the development of GeneRide vectors to durably treat multiple genetic diseases with liver dysfunction.

EXPANSION OF GENERIDE PLATFORM & CAPSID DEVELOPMENT

The unique potential of the GeneRide platform to support development of ther­apies like LB-001 that can target pediatric indications, where other genetic medicines cannot, has been recognized by others in the industry. In January 2020, LogicBio an­nounced a research collaboration with Takeda to further develop LB-301, an in­vestigational therapy for Crigler-Najjar syndrome based on the GeneRide plat­form. In April 2021, LogicBio entered into a research collaboration with Daiichi Sankyo for the development of treatments for two indications based on GeneRide. The agreement also grants Daiichi Sankyo an exclusive option to negotiate to enter into a worldwide license to develop and commercialize LogicBio’s treatments in these two indications. In the same month, LogicBio also entered into a strategic col­laboration with CANbridge Pharmaceuti­cals for an exclusive option to obtain an exclusive license to develop and commercialize LB-001 in Greater China.

As part of this agreement, CANbridge was also granted a worldwide license for certain intellectual property rights, includ­ing those relating to AAV sL65, the first capsid produced based on LogicBio’s sAAVy^TM platform, to develop, manufac­ture and commercialize gene therapy can­didates for the treatment of Fabry and Pompe disease. LogicBio also granted CANbridge options to license sL65 and certain other intellectual property rights for the development and commercialization of two additional gene therapy candidates for the treatment of two additional indica­tions. Similar to the GeneRide platform, sAAVy is uniquely designed to overcome limitations with older-generation AAV tech­nologies by bringing enhanced functional­ity with the potential for increased safety. To design these next-generation AAV cap­sids, LogicBio, together with the Transla­tional Vectorology Research Team at Children’s Medical Research Institute (CMRI), apply innovative genetic and cell and molecular biology techniques, includ­ing bioinformatics, machine learning, and other advanced computational methods.

Based on recent preclinical data, the sAAVy platform shows high potency in a humanized mouse model and in non-human primates compared to widely used benchmark capsids. The sL65 capsid also shows high production yields in suspen­sion HEK293 cells and in bioreactors, meaning it can potentially overcome the current limitations of traditional AAV vec­tors, including high dosage-related toxicity, high manufacturing costs, and low trans­latability from mouse studies to human tri­als. These data were presented at the American Society of Gene and Cell Therapy (ASGCT) meeting in May 2021.⁹

SUMMARY

In developing promising genome ed­iting therapies for any disease or condi­tion, including rare diseases such as MMA, Crigler-Najjar syndrome, HT1, Wilson dis­ease, Fabry and Pompe disease, it is es­sential to work to understand the mechanism of disease and disease path­ways as well as the unmet need and pa­tient experience. Of the 7,000 rare diseases, about 80% have genetic origins, with some caused by mutations in multiple genes (polygenic).¹⁰ Treating these dis­eases is often very complex and presents important considerations regarding vector delivery, manufacturing, and regulatory requirements. Genome editing will be an important technique for treating both monogenic and polygenic diseases and potentially non-genetic diseases in the fu­ture. With the advancement of novel tech­nologies such as GeneRide, there is the promise of developing genome editing therapies that will make a life changing difference for people, at any age, who have few or no treatment options.

REFERENCES

1. Li, H., Yang, Y., Hong, W. et al. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Sig Transduct Target Ther 5, 1 (2020). https://doi.org/10.1038/s41392-019-0089-y.
2. Liu, Z., Chen, O., Wall, J.B.J. et al. Systematic com­parison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7, 2193 (2017). https://doi.org/10.1038/s41598-017-02460-2.
3. Baby’s First Test. (2020). Conditions: Methylmalonic Acidemia (Cobalamin Disorders). https://www.babysfirsttest.org/newborn-screening/conditions/methylmalonic-acidemia-cobal­amin-disorders.
4. National Human Genome Research Institute. (2012, April 20). About Methylmalonic Acidemia. https://www.genome.gov/Genetic-Disorders/MMA-Study-General-Information.
5. Genetic and Rare Diseases Information Center. (2020, Jan 27). Methylmalonic acidemia. https://rarediseases.info.nih.gov/diseases/7033/methylmalonic-acidemia.
6. MedlinePlus. (2020, Oct 29). Methylmalonic acidemia. https://medlineplus.gov/ency/article/001162.htm.
7. Ko, C-W., Gordo, S., Bastille A. et al. (2021, May 11-14). Durable and Efficacious Transgene Expression Driven by GeneRide™ in Liver Injury Models [Confer­ence presentation abstract]. ASGCT 2021 Annual Meeting, Virtual. https://investor.logicbio.com/static-files/247ee792-7dde-4e55-bbb8-3d7c0fa14e9f.
8. Bastille A., Zhang, X., Ramesh, N. et al. (2021, May 11-14). Novel Genome Editing Therapy Improves Disease Phenotype and Survival in a Mouse Model of Methylmalonic Acidemia [Conference presentation abstract]. ASGCT 2021 Annual Meeting, Virtual. https://investor.logicbio.com/static-files/be7513f3-02b0-46c4-9fbd-1fb80c8cf104.
9. Liao, J. (2021, May 11-14). A Novel Liver-Tropic AAV Capsid sL65 Shows Superior Transduction and Effi­cacy in Humanized Mice and Non-Human Primates [Conference presentation]. ASGCT 2021 Annual Meeting, Virtual. https://investor.logicbio.com/static-files/7462e366-2032-41e6-829a-6b8091599c4f.
10. Global Genes. (2021). RARE Facts. https://global­genes.org/rare-facts/.

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Dr. Mariana Nacht is Chief Scientific Officer at LogicBio Therapeutics. She previously served as Chief Scientific Officer and was a founding executive team member of Cereius, where she led a small internal research team and a group of collaborators to develop radiolabeled proteins for the treatment of brain metastases. She has also served in key scientific roles at Vivid Biosciences, Padlock Therapeutics (acquired by Bristol Myers Squibb in 2014), and Avila Therapeutics (acquired by Celgene in 2012). Earlier in her career, she spent a decade working at Genzyme (now Sanofi Genzyme), where she led anti-angiogenesis and oncology target discovery efforts. She earned her BS in Biology from Tufts University and her PhD from the University of Pennsylvania.