CMN Weekly (18 March 2022) - Your Weekly CRISPR Medicine News

Top picks

• Swiss researchers have developed a size-reduced Cas9 prime editor (PE) lacking the RNaseH domain (PE2Δ RnH ) and an intein-split construct (PE2 p.1153) that is suitable for adeno-associated virus (AAV)-mediated delivery into the liver. The authors corrected the disease-causing Pahenu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing as a proof-of-concept. This resulted in average correction efficiencies of 11.1% in neonates and led to therapeutic reduction of blood phenylalanine without inducing detectable off-target mutations or prolonged liver inflammation.
• To treat β-thalassemia, Chinese researchers have reactivated fetal γ-globin expression in human hematopoietic stem/progenitor cells (HSPCs). They did so by using electroporation with bacterial backbone-free minicircle DNA plasmids. Gene editing and reactivation of γ-globin expression was established in erythroblasts derived from unsorted HSPCs after gene editing, and no significant off-target effects were found by deep sequencing.

Research

• Researchers at Jennifer Doudna's lab have published a paper on the bioRxiv preprint server showing that pre-existing histone modifications can lead to higher homology-directed repair (HDR) rates following cuts by CRISPR-Cas9. By fusing a truncated methyltransferase to Cas9, the team transiently decorated chromatin with specific histone modifications while simultaneously cutting with Cas9, leading to increased HDR rates.
• Chinese researchers have used the ability of CRISPR-RfxCas13d to specifically and precisely cleave single-strand RNAs to treat hereditary hearing loss in Bth mice, an animal model for human DFNA36 due to a point mutation in Tmc1. Injection of CRISPR-reagents targeting the Tmc1-mutation into the inner ear of neonatal Bth mice led to a 70% reduction in Tcm1-expression, improved hair cell survival, and significantly enhanced hearing performance in all ages over eight weeks.
• For the first time, a CRISPR-Cas9-based split gene drive system has been tested in a lepidopteran, the diamondback moth (Plutella xylostella). The British researchers noted that although heritable Cas9-mediated germline cleavage and maternal and paternal Cas9 deposition were observed, rates were far lower than for somatic cleavage events. This indicates robust somatic but limited germline activity of Cas9/sgRNA under the control of selected regulatory elements.
• Researchers in the UK have developed a model to predict base editing outcomes using position-specific sequence determinants in two human cell lines. The authors note that base-editing activity is sequence-biased, with the most pronounced effects from nucleotides flanking the target base.
• American researchers have used Cas9 nickase and a single gRNA to effectively remove duplications in the dystrophin gene with no detectable indels. This contrasts with the same strategy using Cas9 nuclease that produced several indels. The approach might open for new gene therapies for Duchenne muscular dystrophy (DMD).
• According to Dutch researchers, a new quantitative kinetic model can predict SpCas9 activity as a function of time, target, and experimental conditions. The model also improves off-target classification and reveals the physical basis of targeting fidelity.
• German researchers have used dCRISPR-Cas9 fusion proteins to achieve persistent gene silencing in mouse embryonic stem cells and HEK293 cells. dCas9 was fused to the transcription repression domain of methyl CpG–binding protein 2 (MeCP2) and a Krüppel-associated box domain (KRABd). Surprisingly, this effect was achieved and even enhanced in DNA (cytosine-5)-methyltransferase depleted cells.
• Researchers in Iran have used CRISPR-Cas9 to generate universal human cells that are not detected by the immune system and can be used for allogenic cell therapies. The researchers used two gRNAs to create a large deletion in the B2M gene and showed that 11% and 22% of cells received genomic changes as homozygous and heterozygous, respectively.
• pH-responsive nanoparticles produced from chitosan-based polymers have been developed to deliver CRISPR-reagents in cancer therapy. The nanoparticles preferentially release their content into cancer cells due to their acidic environment, and the strategy can also be employed to deliver chemotherapeutics.
• Researchers in Spain have used in vivo CRISPR-Cas9 to inhibit lactate dehydrogenase (LDH) in a mouse model of the monogenic disease primary hyperoxalurias (PH). Delivery was performed with AAV8 vectors, and reduced urine oxalate levels and kidney damage without signs of toxicity or off-targets detected.
• Transfection of SpCas9 or an adenine base editor as mRNA mixed with a commercial nucleofector solution results in up to >90% efficient genome editing in human muscle stem cells from many donors regardless of age and gender and without an enrichment step. The German researchers propose mRNA-mediated delivery of CRISPR-Cas9-based tools as a promising and universal approach for taking gene-edited muscle stem cells into clinical application to treat muscle disease.

Clinical trials

• Wugen announces that the first patient has been dosed in a Phase 1/2 trial of WU-CART-007 for the treatment of relapsed or refractory (R/R) T-cell acute lymphoblastic leukaemia (T-ALL)/lymphoblastic lymphoma (LBL). WU-CART-007 is deploying CRISPR-Cas9 gene-editing technology to delete CD7 and the T-cell receptor alpha constant (TRAC), preventing CAR-T cell fratricide and mitigating the risk of graft-versus-host-disease (GvHD).

Industry

• SNIPR BIOME has announced that it will open offices in the US and the UK effective immediately. The new office in London will be led by Chief Intellectual Property Officer Jasper Clube, while Chief Business Officer Stephanie Krebs will lead the office in Boston.
• EdiGene has entered a non-exclusive, worldwide license agreement with Boston Children’s Hospital for intellectual property related to methods and compositions for increasing fetal haemoglobin levels by disrupting BCL11A expression at the genomic level to treat hemoglobinopathies.
• Iovance Biotherapeutics has received approval from FDA for its investigational new drug (IND) application seeking to start clinical studies for its first TALEN-edited tumour infiltrating lymphocyte (TIL) therapy IOV-4001. The company plans to begin a clinical study evaluating IOV-4001 in patients with metastatic melanoma or stage III or IV non-small cell lung cancer (NSCLC) later this year.

Financial reports - all with updates on CRISPR/gene editing programmes

• Gamida Cell reports a net loss of $89.8 million and a cash position of $95.9 million for 2021. Expected milestones are reported from the company's immunotherapy pipeline of potentially curative cell therapies for patients with solid tumours, blood cancers, and other serious blood diseases.
• Verve Therapeutics reports a net loss of $120.3 million and a cash position of $360.4 million for 2021. Recent developments are reported from the company's programmes to treat cardiovascular disease with single-course gene editing medicines.
• Gracell Biotechnologies reports a net loss of $71.2 million and total revenues of $57 million for 2021. In addition, pipeline highlights are reported from the company's gene-edited CAR-T cell therapies.
• Poseida Therapeutics reports a net loss of $125.0 million and total revenue of $31.2 million for 2021. Programme highlights are reported from the company's gene-editing programmes to treat, for example, cancer and haemophilia.
• Precision BioSciences reports a net loss of $30.6 million and total revenues of $115.5 million for 2021. Recent developments and upcoming milestones are reported from the company's many ex vivo and in vivo gene editing programmes.

Detection

• American researchers have used the thermostable Brevibacillus sp. Cas12b to create a test that within 10-30 minutes can determine which variant of Sars-CoV-2 a patient is infected with: alpha, beta, gamma, delta, or omicron. The one-pot detection reaction includes reverse transcription loop-mediated isothermal amplification (RT-LAMP). All reagents are available in a lyophilised version combined with an in-house portable multiplexing device to facilitate dissemination and global implementation of the assay.
• The Crimean-Congo hemorrhagic fever virus (CCHFV) demonstrates broad sequence diversity, making it difficult to detect by CRISPR. Now, Canadian and American researchers have developed a degenerate sequence-based CRISPR diagnostic assay that utilises crRNAs synthesised with mixed nucleotides. The degenerate assay can detect all CCHFV clades effectively.
• An automated centrifugal microfluidic platform for CRISPR-based detection of hepatitis B virus (HBV) is described by Chinese researchers. The platform can separate serum and adsorb, wash, elute and detect DNA using recombinase polymerase amplification (RPA), T7 transcription technology and CRISPR to yield an ultrabright signal that avoids false-negative results.

Reviews

• A paper in Nature Protocols describes methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through the delivery of CRISPR components into living mice. The German authors introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies, and describe applications, ranging from gene editing and cancer modelling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control.
• A review by Chinese researchers discusses the 'endless possibilities for electrochemical nucleic acid sensors' that the CRISPR-Cas-system enables. The study highlights the application of electrochemical biosensors based on CRISPR-Cas to detect and identify cancers, bacteria, and viruses.
• Another Chinese review focuses on integrating CRISPR-Cas with isothermal amplification for point-of-care detection of nucleic acids. The challenges and perspectives in biosensing and clinical applications are analysed.

Opinions and perspectives

• The FDA has published draft guidance for human gene therapy products incorporating human genome editing. According to Fierce Biotech, analysts say that the guidance is 'par for the course' but still important.
• The FDA has also published draft guidance of considerations for developing chimeric antigen receptor (CAR) T cell products. The guidance is expected to increase clarity regarding CMC (chemistry, manufacturing, and controls) in clinical programmes and might help avoid such programmes being delayed or put on hold.
• A podcast from the American Lung Association tries to answer the question "How Close Are CRISPR, Gene Therapies to Lung Disease Breakthroughs?" in a prospective and comprehensive discussion of gene therapy for pulmonary disease between a pair of leading scientists in the field.

News from CRISPR Medicine News

• Monday's feature article reported how researchers at Jennifer Doudna's lab had used DNA origami as a new CRISPR delivery system. Folding DNA into compact structures allows for efficient integration of large genes, and this might lead to new therapies for genetic diseases with many disease-causing mutations and deletions.
• This week's clinical update included news about two gene-edited therapies for cancer. Allogene Therapeutics' ALLO-316 has been granted Fast Track Designation from the FDA to treat advanced kidney cancer. In addition, Intellia Therapeutics' NTLA-5001 has been granted Orphan Drug Designation to treat acute myeloid leukaemia.