An official website of the United States government
Autologous photoreceptor cell replacement is one of the most promising strategies currently being developed for the treatment of patients with inherited retinal degenerative blindness. Induced pluripotent stem cell–derived (iPSC-derived) retinal organoids, which faithfully recapitulate the structure of the neural retina, are an ideal source of transplantable photoreceptors required for these therapies. However, retinal organoids contain other retinal cell types, including bipolar, horizontal, and amacrine cells, which are unneeded and may reduce the potency of the final therapeutic product. Therefore, approaches for isolating fate-committed photoreceptor cells from dissociated retinal organoids are desirable. In this work, we present partial dissociation, a technique that leverages the high level of organization found in retinal organoids to enable selective enrichment of photoreceptor cells without the use of specialized equipment or reagents such as antibody labels. We demonstrate up to 90% photoreceptor cell purity by simply selecting cell fractions liberated from retinal organoids during enzymatic digestion in the absence of mechanical dissociation. Since the presented approach relies on the use of standard plasticware and commercially available current good manufacturing practice–compliant reagents, we believe that it is ideal for use in the preparation of clinical photoreceptor cell replacement therapies.
more »
« less
CGMP Compliant Microfluidic Transfection of Induced Pluripotent Stem Cells for CRISPR-Mediated Genome Editing
Abstract Inherited retinal degeneration is a term used to describe heritable disorders that result from the death of light sensing photoreceptor cells. Although we and others believe that it will be possible to use gene therapy to halt disease progression early in its course, photoreceptor cell replacement will likely be required for patients who have already lost their sight. While advances in autologous photoreceptor cell manufacturing have been encouraging, development of technologies capable of efficiently delivering genome editing reagents to stem cells using current good manufacturing practices (cGMP) are needed. Gene editing reagents were delivered to induced pluripotent stem cells (iPSCs) using a Zephyr microfluidic transfection platform (CellFE). CRISPR-mediated cutting was quantified using an endonuclease assay. CRISPR correction was confirmed via digital PCR and Sanger sequencing. The resulting corrected cells were also karyotyped and differentiated into retinal organoids. We describe use of a novel microfluidic transfection platform to correct, via CRISPR-mediated homology-dependent repair (HDR), a disease-causing NR2E3 mutation in patient-derived iPSCs using cGMP compatible reagents and approaches. We show that the resulting cell lines have a corrected genotype, exhibit no off-target cutting, retain pluripotency and a normal karyotype and can be differentiated into retinal tissue suitable for transplantation. The ability to codeliver CRISPR/Cas9 and HDR templates to patient-derived iPSCs without using proprietary transfection reagents will streamline manufacturing protocols, increase the safety of resulting cell therapies, and greatly reduce the regulatory burden of clinical trials.
more »
« less
- Award ID(s):
- 2134701
- PAR ID:
- 10473245
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Stem Cells
- Volume:
- 41
- Issue:
- 11
- ISSN:
- 1066-5099
- Format(s):
- Medium: X Size: p. 1037-1046
- Size(s):
- p. 1037-1046
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract CRISPR gene editing offers unprecedented genomic and transcriptomic control for precise regulation of cell function and phenotype. However, delivering the necessary CRISPR components to therapeutically relevant cell types without cytotoxicity or unexpected side effects remains challenging. The RALA cell penetrating peptide is an amphiphilic peptide that self‐assembles into nanoparticles through electrostatic interactions with anionic molecules and delivers them across the cell membrane. Given the low cytotoxicity, versatility, and competitive transfection rates of RALA, we aimed to establish this peptide as a new CRISPR delivery system in a wide range of molecular formats across different editing modalities. We report that RALA effectively encapsulated and delivered CRISPR DNAs, RNAs, and ribonucleic proteins (RNPs) to primary mesenchymal stem cells (MSCs), outperforming commercially available reagents. We then used the RALA peptide for the knock‐in and knock‐out of reporter genes into primary MSCs and the transcriptional activation of therapeutically relevant genes. Finally, we demonstrate in vivo gene editing using RALA to knock‐out luciferase and GFP in a reporter mouse model. In summary, we establish RALA as a powerful tool for safer and more effective delivery of CRISPR machinery in multiple cargo formats for a wide range of ex vivo and in vivo gene editing strategies.more » « less
-
Abstract CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.more » « less
-
Abstract A high‐throughput non‐viral intracellular delivery platform is introduced for the transfection of large cargos with dosage‐control. This platform, termed Acoustic‐Electric Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. Intracellular delivery of a wide range of molecule sizes (<1 kDa to 2 MDa) with high efficiency (>90%), cell viability (>80%), and uniform dosages (<60% coefficient of variation (CV)) simultaneously into 1 million cells min−1per single chip is demonstrated. AESOP is successfully applied to two gene editing applications that require the delivery of large plasmids: i) enhanced green fluorescent protein (eGFP) plasmid (6.1 kbp) transfection, and ii) clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9‐mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and single guide RNA (sgRNA). Compared to alternative platforms, this platform offers dosage‐controlled intracellular delivery of large plasmids simultaneously to large populations of cells while maintaining cell viability at comparable delivery efficiencies.more » « less
-
null (Ed.)CRISPR-mediated genome editing has been widely applied in plants to make uncomplicated genomic modifications including gene knockout and base changes. However, the introduction of many genetic variants related to valuable agronomic traits requires complex and precise DNA changes. Different CRISPR systems have been developed to achieve efficient sequence insertion and replacement but with limited success. A recent study has significantly improved NHEJ- and HDR-mediated sequence insertion and replacement using chemically modified donor templates. Together with other newly developed precise editing systems, such as prime editing and CRISPR-associated transposases, these technologies will provide new avenues to further the plant genome editing field.more » « less
