Therapeutic Gene Editing

Making Gene Therapy a Reality Through Real-world Application

Rapidly conduct ex vivo gene modification while eliminating the need to produce viral vectors. Produce healthy, engineered cells with therapeutically relevant levels of gene correction using MaxCyte’s delivery platform – – a non-viral technology designed to meet the stringent demands of clinical use ¬– namely the ability to safely and reproducibly modify primary human cells with high efficiency, minimal cell disturbance, low toxicity concerns, and at the scale required for patient treatment.

  • Established record of clinical gene therapy application for multiple indications
  • High efficiency (co)transfection of DNA, RNA and proteins with minimal cell disturbance
  • Computer-controlled platform for highly reproducible delivery of CRISPR, ZFN, and TALEN components
  • Non-viral platform with lab to commercial manufacturing scalability
  • cGMP-compliant, ISO-certified, CE-marked and supported by a US FDA master file, offering a clear regulatory pathway

 

 

Key Scientific Publications

  • CRISPR-Cas9 Gene Repair of Hematopoietic Stem Cells from Patients with X-linked Chronic Granulomatous Disease. Sci Transl Med. 2017. 9(372).
  • Targeted Gene Addition in Human CD34+ Hematopoietic Cells for Correction of X-linked Chronic Granulomatous Disease. Nature Biotechnol. 2016, 34(4).
  • Clinical Scale Zinc Finger Nuclease-Mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma. Mol. Ther. 2015. 23(8).

 

 

Case Study: Chronic Granulomatous Disease (CGD)

CGD is a genetic disorder that impairs the function of the immune system and leads to recurrent and severe bacterial infections. X- linked CGD (X-CGD), the most prevalent form of CGD, is caused by a mutation in the CYBB gene which encodes a critical component (gp91-phox) of NADPH oxidase, an enzyme that is key for the anti-microbial activity of phagocytes. Correction of the mutated gene within the patients’ own cells via gene editing offers a new curative treatment for X-CGD patients.

In 2015 and again in 2017 MaxCyte received Maryland Stem Cell Research Fund grants to pursue a collaboration with the National Institute of Allergy and Infectious Diseases (NIAID) to develop pre-clinical processes and clinical-scale protocols for CGD and other rare diseases. Most recently a cooperative research and development agreement (CRADA) was announced with NIAID to conduct pre-clinical research evaluating the effectiveness and safety of CRISPR-Cas9 gene editing.

Initial results have demonstrated therapeutic levels of gene correction in the CYBB gene mutation that restored function in long-term engrafted hematopoietic stem cells (HSC) and differentiated neutrophils in cells obtained from individuals with X-CGD. These results were published in Science Translational Medicine in an article entitled “CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease.”

 

High Engraftment Rates of Patient Corrected HSPCs

Engraftment rates in mouse bone marrow for CD34+ HSPCs from Patient 1 (P1) corrected with different concentrations of ssODN (mg/ml), P1 uncorrected (naïve) or healthy donor (HD). Columns show means ± SD; each mouse received 1 million to 3 million corrected P1 CD34+ HSPCs.

 

Long-term Engraftment – 20 Weeks post Transplant

Mouse bone marrow 20 weeks after transplant was sorted for human CD45+ myeloid cells derived from P1 corrected or uncorrected or healthy donor CD34+ HSPCs and stained for gp91phox expression.

 

Restored NOX2 Activity of Engrafted, Gene Corrected Patient Cells

NOX2 activity in human CD45+ cells sorted from mouse bone marrow was determined using the DHR assay after transplant of NSG mice with P1 corrected or uncorrected CD34+ HSPCs or healthy control donor CD34+ HSPCs.

Case Study: Sickle Cell Disease (SCD)

SCD comprises a group of red blood cell disorders associated with mutations within the gene that encodes hemoglobin. Individuals with SCD have abnormal hemoglobin (hemoglobin S – HbS), which results in red blood cells with an altered sickle cell shape. SCD is a life-long illness in which patients experience episodes of severe pain, are more prone to getting infections, may suffer from anemia, and ultimately may develop organ damage due to poor oxygen delivery.

Correction of a faulty hemoglobin gene with SCD patient cells via gene editing offers a new curative treatment. MaxCyte has an ongoing collaboration with the National Heart, Lung and Blood Institute (NHLBI) and National Institute of Allergy and Infectious Diseases (NIAID). The objective of the collaboration is to develop potential curative therapy based on correcting the faulty gene in SCD using MaxCyte’s delivery platform. In contrast, competing approaches focus on either external therapeutic gene addition using viral vectors (now in early clinical trials) or on creation of hereditary persistence of fetal hemoglobin (HPFH) mutations (preclinical research underway). Neither of these approaches impact the level of faulty HbS, which is the target of MaxCyte’s approach and which are at the core of SCD. Studies to date have demonstrated successful CRISPR-induced correction of the mutation behind SCD in more than 30 percent of patient-derived B cells.

 

CRISPR-mediated Homology-directed Repair in Patient Derived Cells

 

SCD patient B-LCL cells were transfected with CRISPR components for integration of HindIII recognition 6-nucleotide sequence. The amplified genomic DNA by PCR was digested with HindIII and analyzed by agarose gel. The density ratio of digested bands to total of parental band and the digested bands was used as integration rate.

 

30% Bi-allelic Correction Rate in SCD Patient and Healthy HSCs

Desired correction detected by sequencing in health HSC and SCD patient B cells. There are four groups of data. The correction of 1) SCD mutation in SCD patient B cells into wild type genotype and 2) wild type sequence in health HSC into SCD mutation, and the integration of HindIII recognition sequence in 3) SCD patient B cells and 4) health HSC. Genomic DNA was amplified after transfection with the appropriate gRNA and oligo either for integration of HindIII recognition sequence or correction. Sequencing analysis demonstrated that ~30% bi-allelic correction rate for both monogenic nucleotide correction and HindIII recognition sequence integration was achieved.

 

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