Engineering memory T cells as a platform for long-term enzyme replacement therapy in lysosomal storage disorders.

Enzymopathy disorders are the result of missing or defective enzymes. Among these enzymopathies, mucopolysaccharidosis type I is a rare genetic lysosomal storage disorder caused by mutations in the gene encoding alpha-L-iduronidase (IDUA), which ultimately causes toxic buildup of glycosaminoglycans (GAGs). There is currently no cure and standard treatments provide insufficient relief to the skeletal structure and central nervous system (CNS).

Development and testing of a versatile genome editing application reporter (V-GEAR) system.

CRISPR-Cas9 and novel cas fusion proteins leveraging specific DNA targeting ability combined with deaminases or reverse transcriptases have revolutionized genome editing. However, their efficacy heavily relies upon protein variants, targeting single guide RNAs, and surrounding DNA sequence context within the targeted loci. This necessitates the need for efficient and rapid screening methods to evaluate these editing reagents and designs.

Engineering Memory T Cells as a platform for Long-Term Enzyme Replacement Therapy in Lysosomal Storage Disorders.

Enzymopathy disorders are the result of missing or defective enzymes. Amongst these enzymopathies, mucopolysaccharidosis type I, is a rare genetic lysosomal storage disorder caused by mutations in the gene encoding alpha-L-iduronidase (IDUA), ultimately causes toxic build-up of glycosaminoglycans (GAGs). There is currently no cure and standard treatments provide insufficient relief to the skeletal structure and central nervous system (CNS).

Precision Enhancement of CAR-NK Cells through Non-Viral Engineering and Highly Multiplexed Base Editing.

Natural killer (NK) cells' unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However, NK cells have demonstrated only moderate responses against cancer in clinical trials and likely require advanced genome engineering to reach their full potential as a cancer therapeutic. Multiplex genome editing with CRISPR/Cas9 base editors (BE) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells.

Generation and characterization of an immunodeficient mouse model of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome, MPS II) is an inherited X-linked recessive disease caused by deficiency of iduronate-2-sulfatase (IDS), resulting in the accumulation of the glycosaminoglycans (GAG) heparan and dermatan sulfates. Mouse models of MPS II have been used in several reports to study disease pathology and to conduct preclinical studies for current and next generation therapies. Here, we report the generation and characterization of an immunodeficient mouse model of MPS II, where CRISPR/Cas9 was employed to knock out a portion of the murine IDS gene on the NOD/SCID/Il2rγ (NSG) immunodeficient background.

Non-invasive intravenous administration of AAV9 transducing iduronate sulfatase leads to global metabolic correction and prevention of neurologic deficits in a mouse model of Hunter syndrome.

Hunter syndrome is a rare x-linked recessive genetic disorder that affects lysosomal metabolism due to deficiency of iduronate-2-sulfatase (IDS), with subsequent accumulation of glycosaminoglycans heparan and dermatan sulfates (GAG). Enzyme replacement therapy is the only FDA-approved remedy and is an expensive life-time treatment that alleviates some symptoms of the disease without neurocognitive benefit. We previously reported successful treatment in a mouse model of mucopolysaccharidosis type II (MPS II) using adeno-associated viral vector serotype 9 encoding human IDS (AAV9.

Neurologic Recovery in MPS I and MPS II Mice by AAV9-Mediated Gene Transfer to the CNS After the Development of Cognitive Dysfunction.

The mucopolysaccharidoses (MPS) are a group of recessively inherited conditions caused by deficiency of lysosomal enzymes essential to the catabolism of glycosaminoglycans (GAG). MPS I is caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA), while MPS II is caused by a lack of iduronate-2-sulfatase (IDS). Lack of these enzymes leads to early mortality and morbidity, often including neurological deficits.

A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.

Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research.

Correction of Fanconi Anemia Mutations Using Digital Genome Engineering.

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk.

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9.

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function-to produce large amounts of antibodies-can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies.

Implication of in Accelerating Tumor Development and Therapeutically Targeting ZNF217-Induced PI3K-AKT Signaling for the Treatment of Metastatic Osteosarcoma.

We previously identified as an oncogenic driver of a subset of osteosarcomas using the () transposon system. Here, we followed up by investigating the genetic role of in osteosarcoma initiation and progression through the establishment of a novel genetically engineered mouse model, assays, orthotopic mouse studies, and paired these findings with preclinical studies using a small-molecule inhibitor. Throughout, we demonstrate that ZNF217 is coupled to numerous facets of osteosarcoma transformation, including proliferation, cell motility, and anchorage independent growth, and ultimately promoting osteosarcoma growth, progression, and metastasis in part through positive modulation of PI3K-AKT survival signaling.

PLX3397 treatment inhibits constitutive CSF1R-induced oncogenic ERK signaling, reduces tumor growth, and metastatic burden in osteosarcoma.

Osteosarcoma (OSA) is a heterogeneous and aggressive solid tumor of the bone. We recently identified the colony stimulating factor 1 receptor (Csf1r) gene as a novel driver of osteosarcomagenesis in mice using the Sleeping Beauty (SB) transposon mutagenesis system. Here, we report that a CSF1R-CSF1 autocrine/paracrine signaling mechanism is constitutively activated in a subset of human OSA cases and is critical for promoting tumor growth and contributes to metastasis.

CRISPR/Cas9-Mediated Genome Engineering of Primary Human B Cells.

The CRISPR/Cas9 system allows for site-specific gene editing and genome engineering of primary human cells. Here we describe methods for gene editing and genome engineering of B cells isolated from human peripheral blood mononuclear cells using CRISPR/Cas9. Editing frequencies of up to 90% and integration rates greater than 60% can be achieved with this method.

ZFN-Mediated In Vivo Genome Editing Corrects Murine Hurler Syndrome.

Mucopolysaccharidosis type I (MPS I) is a severe disease due to deficiency of the lysosomal hydrolase α-L-iduronidase (IDUA) and the subsequent accumulation of the glycosaminoglycans (GAG), leading to progressive, systemic disease and a shortened lifespan. Current treatment options consist of hematopoietic stem cell transplantation, which carries significant mortality and morbidity risk, and enzyme replacement therapy, which requires lifelong infusions of replacement enzyme; neither provides adequate therapy, even in combination. A novel in vivo genome-editing approach is described in the murine model of Hurler syndrome.

Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease.

B cells offer unique opportunities for gene therapy because of their ability to secrete large amounts of protein in the form of antibody and persist for the life of the organism as plasma cells. Here, we report optimized CRISPR/Cas9 based genome engineering of primary human B cells. Our procedure involves enrichment of CD19 B cells from PBMCs followed by activation, expansion, and electroporation of CRISPR/Cas9 reagents.

Dose-Dependent Prevention of Metabolic and Neurologic Disease in Murine MPS II by ZFN-Mediated In Vivo Genome Editing.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by deficiency of iduronate 2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs) in tissues of affected individuals, progressive disease, and shortened lifespan. Currently available enzyme replacement therapy (ERT) requires lifelong infusions and does not provide neurologic benefit. We utilized a zinc finger nuclease (ZFN)-targeting system to mediate genome editing for insertion of the human IDS (hIDS) coding sequence into a "safe harbor" site, intron 1 of the albumin locus in hepatocytes of an MPS II mouse model.

Prevention of Neurocognitive Deficiency in Mucopolysaccharidosis Type II Mice by Central Nervous System-Directed, AAV9-Mediated Iduronate Sulfatase Gene Transfer.

Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a rare X-linked recessive lysosomal disorder caused by defective iduronate-2-sulfatase (IDS), resulting in accumulation of heparan sulfate and dermatan sulfate glycosaminoglycans (GAGs). Enzyme replacement is the only Food and Drug Administration-approved therapy available for MPS II, but it is expensive and does not improve neurologic outcomes in MPS II patients. This study evaluated the effectiveness of adeno-associated virus (AAV) vector encoding human IDS delivered intracerebroventricularly in a murine model of MPS II.