Gene Therapy Potential for Genetic Disorders of Surfactant Dysfunction

Pulmonary surfactant is critically important to prevent atelectasis by lowering the surface tension of the alveolar lining liquid. While respiratory distress syndrome (RDS) is common in premature infants, severe RDS in term and late preterm infants suggests an underlying genetic etiology. Pathogenic variants in the genes encoding key components of pulmonary surfactant including surfactant protein B (SP-B, SFTPB gene), surfactant protein C (SP-C, SFTPC gene), and the ATP-Binding Cassette transporter A3 (ABCA3, ABCA3 gene) result in severe neonatal RDS or childhood interstitial lung disease (chILD). These proteins play essential roles in pulmonary surfactant biogenesis and are expressed in alveolar epithelial type II cells (AEC2), the progenitor cell of the alveolar epithelium. SP-B deficiency most commonly presents in the neonatal period with severe RDS and requires lung transplantation for survival. SFTPC mutations act in an autosomal dominant fashion and more commonly presents with chILD or idiopathic pulmonary fibrosis than neonatal RDS. ABCA3 deficiency often presents as neonatal RDS or chILD. Gene therapy is a promising option to treat monogenic lung diseases. Successes and challenges in developing gene therapies for genetic disorders of surfactant dysfunction include viral vector design and tropism for target cell types. In this review, we explore adeno-associated virus (AAV), lentiviral, and adenoviral (Ad)-based vectors as delivery vehicles. Both gene addition and gene editing strategies are compared to best design treatments for lung diseases resulting from pathogenic variants in the SFTPB, SFTPC, and ABCA3 genes.

The Role of Recombinant AAV in Precise Genome Editing

The replication-defective, non-pathogenic, nearly ubiquitous single-stranded adeno-associated viruses (AAVs) have gained importance since their discovery about 50 years ago. Their unique life cycle and virus-cell interactions have led to the development of recombinant AAVs as ideal genetic medicine tools that have evolved into effective commercialized gene therapies. A distinctive property of AAVs is their ability to edit the genome precisely. In contrast to all current genome editing platforms, AAV exclusively utilizes the high-fidelity homologous recombination (HR) pathway and does not require exogenous nucleases for prior cleavage of genomic DNA. Together, this leads to a highly precise editing outcome that preserves genomic integrity without incorporation of indel mutations or viral sequences at the target site while also obviating the possibility of off-target genotoxicity. The stem cell-derived AAV (AAVHSCs) were found to mediate precise and efficient HR with high on-target accuracy and at high efficiencies. AAVHSC editing occurs efficiently in post-mitotic cells and tissues in vivo. Additionally, AAV also has the advantage of an intrinsic delivery mechanism. Thus, this distinctive genome editing platform holds tremendous promise for the correction of disease-associated mutations without adding to the mutational burden. This review will focus on the unique properties of direct AAV-mediated genome editing and their potential mechanisms of action.

Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants

Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated genome editing is still at the early development stage in plants. In this study, we established an efficient and simplified protoplast-based genome editing platform for CRISPR-Cas RNP delivery, and then evaluated the efficiency, specificity, and temperature sensitivity of six Cas9 and Cas12a proteins. Our results demonstrated that Cas9 and Cas12a RNP delivery resulted in genome editing frequencies (8.7–41.2%) at various temperature conditions, 22°C, 26°C, and 37°C, with no significant temperature sensitivity. LbCas12a often exhibited the highest activities, while AsCas12a demonstrated higher sequence specificity. The high activities of CRISPR-Cas RNPs at 22° and 26°C, the temperature preferred by plant transformation and tissue culture, led to high mutagenesis efficiencies (34.0–85.2%) in the protoplast-regenerated calli and plants with the heritable mutants recovered in the next generation. This RNP delivery approach was further extended to pennycress (Thlaspi arvense), soybean (Glycine max) and Setaria viridis with up to 70.2% mutagenesis frequency. Together, this study sheds light on the choice of RNP reagents to achieve efficient transgene-free genome editing in plants.

A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs

The ultimate goal of technology development in genome editing is to enable precisely targeted genomic changes in any cells or organisms. Here we describe protoplast systems for precise and efficient DNA sequence changes with preassembled Cas9 ribonucleoprotein (RNP) complexes in Arabidopsis thaliana, Nicotiana benthamiana, Brassica rapa, and Camelina sativa. Cas9 RNP-mediated gene disruption with dual gRNAs could reach ∼90% indels in Arabidopsis protoplasts. To facilitate facile testing of any Cas9 RNP designs, we developed two GFP reporter genes, which led to sensitive detection of nonhomologous end joining (NHEJ) and homology-directed repair (HDR), with editing efficiency up to 85 and 50%, respectively. When co-transfected with an optimal single-stranded oligodeoxynucleotide (ssODN) donor, precise editing of the AtALS gene via HDR reached 7% by RNPs. Significantly, precise mutagenesis mediated by preassembled primer editor (PE) RNPs led to 50% GFP reporter gene recovery in protoplasts and up to 4.6% editing frequency for the specific AtPDS mutation in the genome. The rapid, versatile and efficient gene editing by CRISPR RNP variants in protoplasts provides a valuable platform for development, evaluation and optimization of new designs and tools in gene and genomic manipulation and is applicable in diverse plant species.

Targeted Gene Knockouts by Protoplast Transformation in the Moss Physcomitrella patens

Targeted gene knockout is particularly useful for analyzing gene functions in plant growth, signaling, and development. By transforming knockout cassettes consisting of homologous sequences of the target gene into protoplasts, the classical gene targeting method aims to obtain targeted gene replacement, allowing for the characterization of gene functions in vivo. The moss Physcomitrella patens is a known model organism for a high frequency of homologous recombination and thus harbors a remarkable rate of gene targeting. Other moss features, including easy to culture, dominant haploidy phase, and sequenced genome, make gene targeting prevalent in Physcomitrella patens. However, even gene targeting was powerful to generate knockouts, researchers using this method still experienced technical challenges. For example, obtaining a good number of targeted knockouts after protoplast transformation and regeneration disturbed the users. Off-target mutations such as illegitimate random integration mediated by nonhomologous end joining and targeted insertion wherein one junction on-target but the other end off-target is commonly present in the knockouts. Protoplast fusion during transformation and regeneration was also a problem. This review will discuss the advantages and technical challenges of gene targeting. Recently, CRISPR-Cas9 is a revolutionary technology and becoming a hot topic in plant gene editing. In the second part of this review, CRISPR-Cas9 technology will be focused on and compared to gene targeting regarding the practical use in Physcomitrella patens. This review presents an updated perspective of the gene targeting and CRISPR-Cas9 techniques to plant biologists who may consider studying gene functions in the model organism Physcomitrella patens.

Non-GM Genome Editing Approaches in Crops

CRISPR/Cas-based genome editing technologies have the potential to fast-track large-scale crop breeding programs. However, the rigid cell wall limits the delivery of CRISPR/Cas components into plant cells, decreasing genome editing efficiency. Established methods, such as Agrobacterium tumefaciens-mediated or biolistic transformation have been used to integrate genetic cassettes containing CRISPR components into the plant genome. Although efficient, these methods pose several problems, including 1) The transformation process requires laborious and time-consuming tissue culture and regeneration steps; 2) many crop species and elite varieties are recalcitrant to transformation; 3) The segregation of transgenes in vegetatively propagated or highly heterozygous crops, such as pineapple, is either difficult or impossible; and 4) The production of a genetically modified first generation can lead to public controversy and onerous government regulations. The development of transgene-free genome editing technologies can address many problems associated with transgenic-based approaches. Transgene-free genome editing have been achieved through the delivery of preassembled CRISPR/Cas ribonucleoproteins, although its application is limited. The use of viral vectors for delivery of CRISPR/Cas components has recently emerged as a powerful alternative but it requires further exploration. In this review, we discuss the different strategies, principles, applications, and future directions of transgene-free genome editing methods.

Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase

Multidrug-resistant Mycobacterium tuberculosis (Mtb) infection seriously endangers global human health, creating an urgent need for new treatment strategies. Efficient genome editing tools can facilitate identification of key genes and pathways involved in bacterial physiology, pathogenesis, and drug resistance mechanisms, and thus contribute to the development of novel treatments for drug-resistant tuberculosis. Here, we report a two-plasmid system, MtbCBE, used to inactivate genes and introduce point mutations in Mtb. In this system, the assistant plasmid pRecX-NucSE107A expresses RecX and NucSE107A to repress RecA-dependent and NucS-dependent DNA repair systems, and the base editor plasmid pCBE expresses a fusion protein combining cytidine deaminase APOBEC1, Cas9 nickase (nCas9), and uracil DNA glycosylase inhibitor (UGI). Together, the two plasmids enabled efficient G:C to A:T base pair conversion at desired sites in the Mtb genome. The successful development of a base editing system will facilitate elucidation of the molecular mechanisms underlying Mtb pathogenesis and drug resistance and provide critical inspiration for the development of base editing tools in other microbes.

Transgene-free Genome Editing in Plants

Genome editing is widely used across plant species to generate and study the impact of functional mutations in crop improvement. However, transgene integration in plant genomes raises important legislative concerns regarding genetically modified organisms. Several strategies have been developed to remove or prevent the integration of gene editor constructs, which can be divided into three major categories: 1) elimination of transgenic sequences via genetic segregation; 2) transient editor expression from DNA vectors; and 3) DNA-independent editor delivery, including RNA or preassembled Cas9 protein-gRNA ribonucleoproteins (RNPs). Here, we summarize the main strategies employed to date and discuss the advantages and disadvantages of using these different tools. We hope that our work can provide important information concerning the value of alternative genome editing strategies to advance crop breeding.

Targeted Knockout of Eukaryotic Translation Initiation Factor 4E Confers Bymovirus Resistance in Winter Barley

The Eukaryotic Translation Initiation Factor 4E (EIF4E) is a well-known susceptibility factor for potyvirus infections in many plant species. The barley yellow mosaic virus disease, caused by the bymoviruses Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), can lead to yield losses of up to 50% in winter barley. In autumn, the roots of young barley plants are infected by the soil-borne plasmodiophoraceous parasite Polymyxa graminis L. that serves as viral vector. Upon viral establishment and systemic spreading into the upper parts of the plants, yellow mosaics occur as first symptoms on leaves. In the further course of plant development, the disease entails leaf necrosis and increased susceptibility to frost damage. Thanks to the rym4 and rym5 allelic variants of the HvEIF4E gene, more than two thirds of current European winter barley cultivars are resistant to BaYMV and BaMMV. However, several strains of BaYMV and BaMMV have already overcome rym4- and rym5-mediated resistance. Accordingly, new resistance-conferring alleles are needed for barley breeding. Therefore, we performed targeted mutagenesis of the EIF4E gene by Cas9 endonuclease in BaMMV/BaYMV-susceptible winter barley cv. “Igri”. Small insertions were generated, resulting in a shift of the translational reading frame, thereby causing the loss-of-function of EIF4E. The mutations occurred in the homozygous state already in the primary mutants. Their progeny proved invariably homozygous and fully resistant to mechanical inoculation with BaMMV. EIF4E knockout plants showed normal growth habit and produced grains, yet exhibited a yield penalty.

Prime Editing for Inherited Retinal Diseases

Inherited retinal diseases (IRDs) are chronic, hereditary disorders that lead to progressive degeneration of the retina. Disease etiology originates from a genetic mutation—inherited or de novo—with a majority of IRDs resulting from point mutations. Given the plethora of IRDs, to date, mutations that cause these dystrophies have been found in approximately 280 genes. However, there is currently only one FDA-approved gene augmentation therapy, Luxturna (voretigene neparvovec-rzyl), available to patients with RPE65-mediated retinitis pigmentosa (RP). Although clinical trials for other genes are underway, these techniques typically involve gene augmentation rather than genome surgery. While gene augmentation therapy delivers a healthy copy of DNA to the cells of the retina, genome surgery uses clustered regularly interspaced short palindromic repeats (CRISPR)-based technology to correct a specific genetic mutation within the endogenous genome sequence. A new technique known as prime editing (PE) applies a CRISPR-based technology that possesses the potential to correct all twelve possible transition and transversion mutations as well as small insertions and deletions. EDIT-101, a CRISPR-based therapy that is currently in clinical trials, uses double-strand breaks and nonhomologous end joining to remove the IVS26 mutation in the CEP290 gene. Preferably, PE does not cause double-strand breaks nor does it require any donor DNA repair template, highlighting its unparalleled efficiency. Instead, PE uses reverse transcriptase and Cas9 nickase to repair mutations in the genome. While this technique is still developing, with several challenges yet to be addressed, it offers promising implications for the future of IRD treatment.