Gene Correction Research Articles

Challenges in Gene Therapy for Somatic Reverted Mosaicism in X-Linked Combined Immunodeficiency by CRISPR/Cas9 and Prime Editing.

X-linked severe combined immunodeficiency (X-SCID) is a primary immunodeficiency that is caused by mutations in the interleukin-2 receptor gamma (IL2RG) gene. Some patients present atypical X-SCID with mild clinical symptoms due to somatic revertant mosaicism. CRISPR/Cas9 and prime editing are two advanced genome editing tools that paved the way for treating immune deficiency diseases. Prime editing overcomes the limitations of the CRISPR/Cas9 system, as it does not need to induce double-strand breaks (DSBs) or exogenous donor DNA templates to modify the genome. Here, we applied CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs) and prime editing methods to generate an in vitro model of the disease in K-562 cells and healthy donors' T cells for the c. 458T>C point mutation in the IL2RG gene, which also resulted in a useful way to optimize the gene correction approach for subsequent experiments in patients' cells. Both methods proved to be successful and were able to induce the mutation of up to 31% of treated K-562 cells and 26% of treated T cells. We also applied similar strategies to correct the IL2RG c. 458T>C mutation in patient T cells that carry the mutation with revertant somatic mosaicism. However, both methods failed to increase the frequency of the wild-type sequence in the mosaic T cells of patients due to limited in vitro proliferation of mutant cells and the presence of somatic reversion. To the best of our knowledge, this is the first attempt to treat mosaic cells from atypical X-SCID patients employing CRISPR/Cas9 and prime editing. We showed that prime editing can be applied to the formation of specific-point IL2RG mutations without inducing nonspecific on-target modifications. We hypothesize that the feasibility of the nucleotide substitution of the IL2RG gene using gene therapy, especially prime editing, could provide an alternative strategy to treat X-SCID patients without revertant mutations, and further technological improvements need to be developed to correct somatic mosaicism mutations.

Multiplex HDR for disease and correction modeling of SCID by CRISPR genome editing in human HSPCs

Severe combined immunodeficiency (SCID) is a group of disorders caused by mutations in genes involved in the process of lymphocyte maturation and function. CRISPR-Cas9 gene editing of the patient's own hematopoietic stem and progenitor cells (HSPCs) exvivo could provide a therapeutic alternative to allogeneic hematopoietic stem cell transplantation, the current gold standard for treatment of SCID. To eliminate the need for scarce patient samples, we engineered genotypes in healthy donor (HD)-derived CD34+ HSPCs using CRISPR-Cas9/rAAV6 gene-editing, to model both SCID and the therapeutic outcomes of gene-editing therapies for SCID via multiplexed homology-directed repair (HDR). First, we developed a SCID disease model via biallelic knockout of genes critical to the development of lymphocytes; and second, we established a knockin/knockout strategy to develop a proof-of-concept single-allelic gene correction. Based on these results, we performed gene correction of RAG2-SCID patient-derived CD34+ HSPCs that successfully developed into CD3+ Tcells with diverse TCR repertoires in an invitro Tcell differentiation platform. In summary, we present a strategy to determine the optimal configuration for CRISPR-Cas9 gene correction of SCID using HD-derived CD34+ HSPCs, and the feasibility of translating this gene correction approach in patient-derived CD34+ HSPCs.

LMNA Co-Regulated Gene Expression as a Suitable Readout after Precise Gene Correction.

LMNA-related muscular dystrophy is an autosomal-dominant progressive disorder caused by mutations in LMNA. LMNA missense mutations are becoming correctable with CRISPR/Cas9-derived tools. Evaluating the functional recovery of LMNA after gene editing bears challenges as there is no reported direct loss of function of lamin A/C proteins in patient-derived cells. The proteins encoded by LMNA are lamins A/C, important ubiquitous nuclear envelope proteins but absent in pluripotent stem cells. We induced lamin A/C expression in induced pluripotent stem cells (iPSCs) of two patients with LMNA-related muscular dystrophy, NM_170707.4 (LMNA): c.1366A > G, p.(Asn456Asp) and c.1494G > T, p.(Trp498Cys), using a short three-day, serum-induced differentiation protocol and analyzed expression profiles of co-regulated genes, examples being COL1A2 and S100A6. We then performed precise gene editing of LMNA c.1366A > G using the near-PAMless (PAM: protospacer-adjacent motif) cytosine base editor. We show that the mutation can be repaired to 100% efficiency in individual iPSC clones. The fast differentiation protocol provided a functional readout and demonstrated increased lamin A/C expression as well as normalized expression of co-regulated genes. Collectively, our findings demonstrate the power of CRISPR/Cas9-mediated gene correction and effective outcome measures in a disease with, so far, little perspective on therapies.

Advances in Gene Therapy Techniques to Treat LRRK2 Gene Mutation.

Leucine-rich repeat kinase 2 (LRRK2) gene mutation is an autosomal dominant mutation associated with Parkinson's disease (PD). Among LRRK2 gene mutations, the LRRK2 G2019S mutation is frequently involved in PD onset. Currently, diverse gene correction tools such as zinc finger nucleases (ZFNs), helper-dependent adenoviral vector (HDAdV), the bacterial artificial chromosome-based homologous recombination (BAC-based HR) system, and CRISPR/Cas9-homology-directed repair (HDR) or adenine base editor (ABE) are used in genome editing. Gene correction of the LRRK2 G2019S mutation has been applied whenever new gene therapy tools emerge, being mainly applied to induced pluripotent stem cells (LRRK2 G2019S-mutant iPSCs). Here, we comprehensively introduce the principles and methods of each programmable nuclease such as ZFN, CRISPR/Cas9-HDR or ABE applied to LRRK2 G2019S, as well as those of HDAdV or BAC-based HR systems used as nonprogrammable nuclease systems.

Enabling Precision Medicine with CRISPR-Cas Genome Editing Technology: A Translational Perspective.

Genome editing technologies, particularly CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR) associated nucleases), are redefining the boundaries of therapeutic gene therapy. CRISPR-Cas is a robust, straightforward, and programmable genome editing tool capable of mediating site-specific DNA modifications. The rapid advancements from discovery to clinical adaptation have expanded the therapeutic landscape to treat genetically defined diseases. Together with the technical developments in human DNA and RNA sequencing, CRISPR-directed gene therapy enables a new era to realize precision medicine where pathogenic mutations underlying monogenic disorders can potentially be corrected. Also, protective or therapeutic genomic alterations can be introduced as preventative or curative therapy. Despite its high therapeutic potential, CRISPR-Cas´ clinical translation is still in its infancy and is highly dependent on its efficiency, specificity in gene corrections, and cell-specific delivery. Therefore, this chapter focuses on the challenges and opportunities the CRISPR-Cas toolbox offers together with delivery vehicles to realize its use for therapeutic gene editing. Furthermore, we discuss the obstacles the CRISPR-Cas system faces for successful clinical translation and summarize its current clinical progress.

Osteoclast rich osteopetrosis due to defects in the TCIRG1 gene.

Discovery that mutations in TCIRG1 (also known as Atp6i) gene are responsible for most instances of autosomal recessive osteopetrosis (ARO) heralded a new era for comprehension and treatment of this phenotypically heterogeneous rare bone disease. TCIRG1 encodes the a3 subunit, an essential isoform of the vacuolar ATPase proton pump involved in acidification of the osteoclast resorption lacuna and in secretory lysosome trafficking. TCIRG1 defects lead to inefficient bone resorption by nonfunctional osteoclasts seen in abundance on bone marrow biopsy, delineating this ARO as 'osteoclast-rich'. Presentation is usually in early childhood and features of extramedullary haematopoiesis (hepatosplenomegaly, anaemia, thrombocytopenia) due to bone marrow fibrosis, and cranial nerve impingement (blindness in particular). Impaired dietary calcium uptake due to high pH causes the co-occurrence of rickets, described as "osteopetrorickets". Osteoclast dysfunction leads to early death if untreated, and allogeneic haematopoietic stem cell transplantation is currently the treatment of choice. Studies of patients as well as of mouse models carrying spontaneous (the oc/oc mouse) or targeted disruption of Atp6i (TCIRG1) gene have been instrumental providing insight into disease pathogenesis and development of novel cellular therapies that exploit gene correction.

CRISPR-Cas9 gene editing and human diseases.

CRISPR/Cas-9 mediated genome editing has recently emerged as a potential and innovative technology in therapeutic development and biomedical research. Several recent studies have been performed to understand gene modification techniques in obtaining effective ex vivo results. Generally, the disease targets for gene correction will be in specific organs, so understanding the complete potential of genomic treatment methods is essential. From such a perspective, the present review revealed the significant importance of the CRISPR/ Cas9 delivery system. Both the promising gene-editing delivery systems, such as synthetic (non-viral) and viral vector systems are discussed in this review. In addition, this paper attempted to summarize the tissue-specific and organ-specific mRNA delivery systems that provide possible research information for future researchers. Further, the major challenges of the CRISPR/Cas9 system, such as off-target delivery, immunogenicity, and limited packaging, were also elucidated. Accordingly, this review illustrated a wide range of clinical applications associated with the efficient delivery of CRISPR/ Cas9 gene-editing. Moreover, this article emphasizes the role of the CRISPR/Cas9 system in treating Intra Cerebral haemorrhage (ICH), thereby suggesting future researchers to adopt more clinical trials on this breakthrough delivery system.

Description of CRISPR-Cas9 development and its prospects in human papillomavirus-driven cancer treatment.

Human papillomaviruses (HPVs) have been recognized as the etiologic agents of various cancers and are called HPV-driven cancers. Concerning HPV-mediated carcinogenic action, gene therapy can cure cancer at the molecular level by means of the correction of specific genes or sites. CRISPR-Cas9, as a novel genetic editing technique, can correct errors in the genome and change the gene expression and function in cells efficiently, quickly, and with relative ease. Herein, we overviewed studies of CRISPR-mediated gene remedies for HPV-driven cancers and summarized the potential applications of CRISPR-Cas9 in gene therapy for cancer.

268 ABE8e adenine base editor corrects COL7A1 nonsense mutation efficiently and safely

Base editing, the introduction of a single base correction in genomic DNA, has the potential to treat genetic skin diseases such as recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by mutations in the COL7A1 gene and type VII collagen (C7) deficiency. Hitherto, base editing has been harnessed to correct disease-causing mutations but a reproducible platform for efficient gene correction and a safe approach for clinical translation remain elusive. Adenine base editors (ABEs) convert A-T base pairs to G-C base pairs without requiring double-stranded DNA breaks or donor DNA templates.

ABE8e adenine base editor precisely and efficiently corrects a recurrent COL7A1 nonsense mutation

Base editing introduces precise single-nucleotide edits in genomic DNA and has the potential to treat genetic diseases such as the blistering skin disease recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by mutations in the COL7A1 gene and type VII collagen (C7) deficiency. Adenine base editors (ABEs) convert A-T base pairs to G-C base pairs without requiring double-stranded DNA breaks or donor DNA templates. Here, we use ABE8e, a recently evolved ABE, to correct primary RDEB patient fibroblasts harboring the recurrent RDEB nonsense mutation c.5047 C > T (p.Arg1683Ter) in exon 54 of COL7A1 and use a next generation sequencing workflow to interrogate post-treatment outcomes. Electroporation of ABE8e mRNA into a bulk population of RDEB patient fibroblasts resulted in remarkably efficient (94.6%) correction of the pathogenic allele, restoring COL7A1 mRNA and expression of C7 protein in western blots and in 3D skin constructs. Off-target DNA analysis did not detect off-target editing in treated patient-derived fibroblasts and there was no detectable increase in A-to-I changes in the RNA. Taken together, we have established a highly efficient pipeline for gene correction in primary fibroblasts with a favorable safety profile. This work lays a foundation for developing therapies for RDEB patients using ex vivo or in vivo base editing strategies.

Characterization of Hemoglobin Variants Due to in-Frame Deletions in the β-Globin Gene from CRISPR/Cas9 Gene Correction for Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene (HBB). CRISPR/Cas9 based gene correction approaches rely on inducing a double-strand break (DSB) at or near the sickle mutation, which subsequently results in gene correction via the homology-directed repair (HDR) or insertions/deletions (Indels) via non-homologous end joining (NHEJ). Frameshift Indels induce a premature stop codon and disrupt HBB production. Indels that preserve the reading frame may yield structurally abnormal β-globin chain with altered amino acid sequences and protein structures that can affect the globin chain stability, leading to thalassemia of uncertain pathogenic significance. The high rate and diversity of in-frame Indels as an unintended by-product of gene correction therapy confers a challenge for predicting therapeutic outcomes when it is not possible to frame the clinical behavior of hemoglobin (Hb) variants. Here we provided the molecular and structural characterization of the most commonly identified Hb variants due to CRISPR-induced in-frame deletions and proposed a putative pathogenic mechanism for the observed phenotype using human umbilical cord-derived erythroid progenitor cells (HUDEP2). Methods: HUDEP2 clonal cell lines carrying mutated Hb variants from in-frame deletions on HBB exon1 introduced by R-66S and R-02 CRISPR sgRNAs currently in use in preclinical and clinical studies for correcting sickle mutation, respectively, were established, and clonal genotypes validated by next-generation sequencing and droplet digital PCR based copy number assay. The Hb variants derived from R-66S include p.Val7_Ser10del (-12bp), p.Val7del (-3bp), p.Glut8del (-3bp), p.Glut8_Lys9del (-6bp), pVal7Thr13del (-21bp), p.His3_Lys9delinsGln (-18bp), and p.Thr5_Lys18del (-42bp), in the order of allelic abundance observed in the bulk population of edited cells. The Hb variants derived from R-02 include p.Val12_Ala14del (-9bp), p.Val12_Thr13del (-6bp), p.Ala14del (-3bp), p.Thr13_Gly17del (-15bp), and p.Thr13_Val19delinsMet (-18bp). Selected ten clonal cell lines carrying a unique combination of genotypes and the parent HUDEP2 were cultured for expansion in serum-free media for one week. All clones were subsequently subjected to erythroid differentiation media for ten days. Samples were collected on days 4, 7, and 10 of differentiation for analysis via high-performance liquid chromatography (HPLC) using an ionic column to access the Hb profile and spectrophotometry via a Hb assay kit to assess the total Hb quantity. Results: HPLC analysis demonstrated a significant qualitative variation in Hb profile in HUDEP2 clones carrying different Hb variants. p.Val7del is Hb Leiden, and the rest of Hb variants are not listed in the HbVar database. In clones carrying Hb Leiden in homozygous or heterozygous states, Hb Leiden eluted in the position of sickle Hb (HbS). A low percentage of multiple abnormal Hb X fractions was observed in all clones carrying Hb variants compared to parent HUDEP2, which hinted at the presence of unstable Hb variants or alpha precipitates. Levels of fetal Hb (HbF) increased in mutant clones, suggesting globin chain imbalance and hematopoietic stress. Clones missing multiple amino acids showed ineffective erythropoiesis and cell death by day 4 of differentiation, most likely exacerbated by the relative excess of the α-chains. Discussion: We found that unintended gene modifications can significantly impact Hb quality and quantity, reinforcing concerns about the safety of gene-editing therapy for SCD. In patients, cells carrying in-frame deletions in the β-globin chain could produce an unstable Hb and may contribute to a β-thalassemia-like phenotype. More work is required to further assess the consequences of the diverse Hb variants in combination with other unintended modifications that disrupt HBB in a bulk population of gene-edited cells. Detecting, profiling, and reducing these unintended modifications remains a priority to increase the efficacy and safety of gene-editing therapy for treating SCD or β-thalassemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clonal Dynamics of HDR-Edited HSPCs Targeting the CD33 Locus in Rhesus Macaques

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. Loss or damage of LT-HSCs with resultant oligoclonal hematopoiesis can increase the risk of malignant transformation or long-term hematopoietic and immune dysfunction. HDR has adverse impacts on LT-HSC function as revealed by ex vivo studies and murine xenograft models, likely due to p53 activation, intracellular innate immune activation, and/or other pathways. However, the impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not previously been studied. We tracked the clonal dynamics of CRISPR/HDR-edited barcoded HSPCs following autologous transplantation in rhesus macaques (RMs). HDR-favored gRNAs targeting the CD33 gene locus and an ssODN containing a 6 bp barcode ID and 14 bp random barcode sequences and flanked by CD33 homology arms were designed. Autologous CD34+ cells were electroporated with the Cas9 RNP and HDR cassette in the presence of i53 mRNA to inhibit p53-mediated cell death and reinfused into animal 1 following myeloablation. In animal 1, CD33 KO neutrophils were prevalent early following engraftment and then rapidly decreased over the next two months. Similar to CD33 expression level, we revealed that HDR knock-ins as well as total indel frequency drastically decreased following transplantation. This was in marked contrast to macaques transplanted with CD34+ HSPCs edited at the CD33 locus only for NHEJ, without inclusion of an HDR cassette (Kim et al, Cell, 2018, and Figure 1 below). We next performed a competitive transplantation comparing clonal dynamics of edited cells to barcoded lentiviral (LV)-transduced cells in two macaques. For each animal, CD34+ cells were fractionated into 2 aliquots, and half were transduced with a barcoded GFP-expressing LV vector and half electroporated with the Cas9 RNP and HDR cassette with higher concentrations of i53 mRNA to enhance protection of CRISPR edited LT-HSCs. The two cell populations were combined and reinfused into myeloablated animals. CD33 KO cells in both animals decreased rapidly, however the higher concentration of i53 mRNA facilitated a less steep reduction in CD33 total indel frequency in vivo over time. However, HDR in these two animals also markedly decreased, similar to the pattern in animal 1. In contrast, LV-transduced GFP+ cell engraftment stabilized at 20% after 2 months. We retrieved embedded barcodes from CRISPR/HDR cells and revealed that a diversity of HDR clones contributed to early hematopoietic reconstitution, with restriction to a much smaller set of long-term clones at steady state, proportional in the 3 animals to the level of HDR overall. The diversity of HDR-edited contributing clones was far less than LV-transduced contributing clones long-term post-transplantation. In conclusion, CRISPR/HDR edited cells were found to decrease rapidly after autologous transplantation in rhesus macaques despite substantial gene editing assessed in the infusion product prior to infusion, in contrast to much more stable LV-transduced cells. Clonality of HDR-edited cells drastically shrank at early stage and then relied on several dominant clones, which was somewhat improved by use of high dose i53 mRNA during editing. It will be important to find improved approaches to retain LT-HSC functional activity during the HDR editing process, or explore targeted gene correction approaches such as base or PRIME editing that may better preserve HSC activity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Development of a β-Globin Gene Replacement Strategy As a β-Thalassemia Therapy

Background: β-thalassemia is a genetic disorder with high unmet need worldwide. Over 300 genetic mutations that result in the reduction, or loss, of β-globin (HBB) expression have been characterized to date. Patients with thalassemic mutations in both HBB alleles often experience severe anemia resulting from ineffective erythropoiesis due to the loss of adult hemoglobin (HbA) tetramers (α2β2) and imbalanced excess of α-globin chains. One challenge is creating a genome-editing strategy that replaces the diversity of disease-causing mutations in the HBB gene while restoring physiological HbA expression. Therefore, a gene-editing platform using homology-directed repair (HDR) that could replace the entire HBB gene in situ and under the control of normal regulatory elements would be an ideal strategy. Methods: CD34+ hematopoietic stem and progenitor cells (HSPCs) were edited using high-fidelity Cas9, HBB guide RNA, and different AAV6 DNA donor templates and then differentiated into red blood cells in vitro. We evaluated HBB expression in 39 AAV6 donors engineered with heterologous introns and different polyadenylation signals first using a flow cytometry screening assay and later HPLC. Candidates that met or exceeded wild-type HBB expression were moved forward for further comparison. Next, we edited HSPCs with the 8 highest expressing AAV6 donors, performed in vitro differentiation, and evaluated HDR frequencies by droplet digital polymerase chain reaction (ddPCR) and HbA formation by HPLC. The candidate that demonstrated the highest conversion of donor knock-in to HbA was further engineered to boost expression and reduce construct size. Two final candidates were moved forward to further characterize their ability to rescue loss of HbA in CD34+ HSPCs isolated from patients with sickle cell disease (SCD) at the protein and RNA level. Additionally, we performed DNA analysis on the gene-edited cells using ddPCR and a targeted PCR-free long-range sequencing approach. Results: Adding heterologous introns to the HBB coding sequence significantly increased HBB expression relative to the no-intron construct containing only coding DNA. The results from our flow cytometry screen and HPLC analysis were positively correlated, and the top DNA donors with heterologous introns resulted in physiologic HbA expression. Using this gene-replacement strategy, we achieved HDR frequencies of up to 40% in CD34+ HSPCs. Further optimization resulted in a smaller DNA template, maintenance of HbA conversion rates, and a lower rate of unintended recombinations. Finally, using HSPCs from patients with SCD as a therapeutically relevant model, we found, by HPLC and RNA sequencing, the optimized DNA donor can successfully replace the entire nonfunctional HBB gene, thereby restoring physiologic HbA expression. Future experiments are investigating in vitro gene correction in cells isolated from patients with β-thalassemia and additional studies evaluating in vivo stem cell repopulation capacity of gene-edited HSPCs. Conclusion: We developed an HBB AAV6 donor engineered with heterologous introns that demonstrates efficient HBB gene replacement and HbA rescue in the erythroid progeny of CD34+ HSPCs isolated from patients with SCD, offering a potential differentiated approach for treating β-thalassemia.

Highly Efficient & Specific Repair of MAGT1 Mutation in Xmen Patient T Cells and Hematopoietic Stem Cells

Introduction: X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia (XMEN) is a congenital glycosylation disorder with combined immune deficiency due to Magnesium Transporter 1 (MAGT1) mutations. Defective glycosylation of Natural Killer Group 2D (NKG2D), an activating receptor expressed on CD8 and NK cells critical for cytotoxicity, results in EBV lymphoproliferative disease and an inverted CD4:CD8 ratio in many XMEN patients. Oral magnesium supplementation for XMEN patients was not helpful, and hematopoietic stem cell transplants (HSCT) are complicated by high mortality. CRISPR-Cas9-AAV inserts MAGT1 specifically at endogenous locus but relies on double strand DNA breaks and adeno-associated virus donor that triggers DNA damage response. To address this, we evaluated base editing approach to repair a MAGT1 mutation that affects multiple patients with XMEN disease. We observed highly efficient mutation repair of XMEN T cells (75.5%) and hematopoietic stem cells (71.9%) to restore MAGT1 function and persistence of gene correction following transplant into immunodeficient mice. Materials and methods: Peripheral blood hematopoietic stem cells (HSPCs, CD34+) and mononuclear cells (PBMC) were collected from XMEN patients and healthy donors (HD) after G-CSF and plerixafor mobilization. PBMCs were stimulated (anti-CD3/anti-CD28, IL-2 (100 IU/mL), in ImmunoCult™-XF T cell expansion medium for 5-7 days prior to editing. HSPCs were cultured in StemSpan™ SFEM II Stem Cell Factor, Thrombopoietin, Fms-like tyrosine kinase 3 ligand (all 100ng/mL) and UM171 for two days prior to editing. Base editing is performed by electroporation (MaxCyte Inc.) of cells with ABE8e SpRY (CELLSCRIPT LLC.) and synthetic guide (Synthego) RNAs. For in vitro killing activity, T cells were co-cultured with EBV-B cells at a ratio of 1:1 for 48 hours and analyzed for apoptosis (Apoptracker Green+ 7AAD-), necrosis (Apoptracker Green+ 7AAD+) and degranulation (CD8+ CD107a+). HSPCs were transplanted into mice two days after EP and harvested 17-20 weeks after transplant. Results:Base editing of XMEN T cells: Flow cytometric analysis 2 days post BE show restored expression of biomarker NKG2D in XMEN CD8+ T cells (75.5%) compared to HD (93.2%) and naïve XMEN (2.39%). CD8+ cytotoxic activity against EBV-B cells was restored in BE XMEN patient cells; as indicated by increased apoptosis (BE: 6.78%, naïve: 4.82%, HD: 8.17%), necrosis (BE: 9.35%, naïve: 4.54%, HD: 7.19%), and degranulation (BE: 22.80%, naïve: 15.50%, HD: 21.40%), compared to naïve MAGT1 CD8+ T cells.In an in vivo tumor model, BE XMEN T cells injected into mice with induced luminescent EBV-B cell tumors lysed tumors and reduced average bioluminescence flux (photons/sec) (7 weeks) (8.23 × 107 ± 1.06 × 108) compared to naïve T cells (3.39 × 109 ± 2.22 × 109 and HD T cells (1.45 × 109 ± 2.22 × 109). Base editing of HSPCs: Immunodeficient NSGS pups transplanted with BE XMEN HSPCs engrafted and differentiated normally into lymphocytes with a normalized CD4:CD8 ratio in spleen (BE: 0.79 ± 0.14, naïve: 3.45 ± 1.1, HD: 1.3 ± 0.40) and peripheral blood (BE: 2.16 ± 0.84, naïve: 5.56 ± 2.32, HD: 1.79 ± 0.70) (Fig 1a). Importantly, restoration of biomarker NKG2D expression selectively on CD8+ T cells in spleen (BE: 80.5% ± 8.61%, naïve: 3.63% ± 1.45%, HD: 68.9% ± 10.32%) and peripheral blood (BE: 86.1% ± 2.88%, naïve: 2.25% ± 1.45%, HD: 62.6% ± 12.44%) (Fig 1b) confirmed correction of MAGT1 function in the glycosylation of critical NKG2D protein. High throughput targeted sequencing of on-target locus shows minimal bystander edits and additional off-target analysis is ongoing. Conclusion: Our data show highly efficient and specific base editing repair of MAGT1 mutation in XMEN T lymphocytes and CD34+ HSPCs. BE XMEN T cells increased cytotoxic activity in vitro and killing of tumor cells in mice. In vivo studies with mice transplanted with BE CD34+ HSPCs also confirm long term engraftment and differentiation into CD8+ T cells with restored NKG2D necessary for activation and cytolytic activity of CD8+ T and NK cells. The highly efficient and specific correction with base editing holds great promise for gene therapy for XMEN disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Unveiling the Biological Role of Peripheral Blood Human Circulating Hematopoietic Stem and Progenitor Cells

Although most hematopoietic stem/progenitor cells (HSPC) reside in the bone marrow (BM), few circulating HSPC (cHSPC) can be also found in the peripheral blood (PB) at steady state. The biological role and relationship with BM-resident counterpart in humans remain still not fully elucidated. In the present study, we phenotypically characterized cHSPC composition by applying multi-parametric flow cytometry on 110 PB and, as control, 48 BM samples of healthy donors (HD) of different age groups. These analyses were combined with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and ad hoc designed in vitro and in vivo assays to investigate the transcriptional and functional properties of steady-state cHSPC subpopulations with respect to BM counterpart. Moreover, to study circulating vs. resident HSPC differentiation potential in vivo in humans, we exploited integration site (IS) clonal tracking of cHSPC, BM HSPC and mature PB lineages isolated from patients treated with autologous HSPC-gene therapy (GT) at late follow-ups post-treatment (>2 years), once steady-state hematopoiesis is established. We observed that cHSPC show a progressive reduction in number during aging and a different composition than BM counterpart, with Multi Lymphoid Progenitors (MLP) displaying the highest PB circulation propensity. cHSPC were endowed with BM homing capability and multilineage differentiation potential both in vitro and in vivo, showing an early skewing toward erythroid differentiation after transplantation. Moreover, we detected a reduced long-term human cell engraftment in cHSPC- than BM HSPC-transplanted mice. This latter finding might be explained by the low primitive Hematopoietic Stem Cell (HSC) content and the transcriptional pre-activated state observed in steady-state PB HSC, as assessed in our single-cell RNA-sequencing analyses. Indeed, applying CITE-seq on PB- and BM-derived HSPC, we identified a unique transcriptional profile of both primitive and lineage-committed cHSPC subpopulations, characterized by lower replicative, metabolic and transcriptional activity, but increased differentiation-, adhesion- and immune response-priming than BM counterpart. Furthermore, we observed that PB phenotypic primitive HSC and Multipotent Progenitors (MPP) and Committed Myeloid Progenitors (CMP) were transcriptionally and functionally skewed toward erythroid lineages as compared to BM-resident counterpart. Finally, since upon gene correction each HSPC and its progeny became univocally marked by distinct IS, we exploited IS analyses from 5 patients treated with HSPC-GT at >2 years after GT to investigate the steady-state hematopoietic output of PB and BM HSPC in vivo in humans. In line with the enrichment of lymphoid transcriptional signature observed in trafficking HSPC, we found a higher IS sharing between PB mature lymphoid compartment and steady-state PB-derived HSPC. Moreover, a substantial fraction of trafficking HSPC displayed an enriched expression of the gene signature associated with thymus seeding progenitor type 1 (TSP1), a group of low-cycling immature lymphoid progenitors with thymus-emigrant properties. Although at lower level with respect to PB cells, we detected a TSP1 signature also in BM dataset, thus suggesting that few thymus-emigrant lymphoid progenitors originate in the BM and preferentially egress into the PB in order to seed lymphoid organs. Altogether, our findings indicate PB trafficking HSPC as a peculiar steady-state reservoir of low-cycling, pre-activated hematopoietic progenitors, which are endowed with BM homing and repopulation potential. The enrichment of phenotypic and functional erythroid-committed progenitors in cHSPC suggest a supportive role of these cells in sustaining extramedullary erythropoiesis. Furthermore, our results of IS analyses combined with the higher expression of TSP1 signature in PB vs. BM HSPC indicate the key function of steady-state trafficking HSPC in the seeding of the thymus, with the aim of locally differentiating into lymphoid progeny. Overall, our work represents one of the most comprehensive studies on cHSPC, unveiling fundamental insights on their biological role in humans.

Enhancement of Gene Editing and Base Editing with Therapeutic Ribonucleoproteins through In Vivo Delivery Based on Absorptive Silica Nanoconstruct.

Key to the widespread and secure application of genome editing tools is the safe and effective delivery of multiple components of ribonucleoproteins (RNPs) into single cells, which remains a biological barrier to their clinical application. To overcome this issue, a robust RNP delivery platform based on a biocompatible sponge-like silica nanoconstruct (SN) for storing and directly delivering therapeutic RNPs, including Cas9 nuclease RNP (Cas9-RNP) and base editor RNP (BE-RNP) is designed. Compared with commercialized material such as lipid-based methods, up to 50-fold gene deletion and 10-fold base substitution efficiency is obtained with a low off-target efficiency by targeting various cells and genes. In particular, gene correction is successfully induced by SN-based delivery through intravenous injection in an in vivo solid-tumor model and through subretinal injection in mouse eye. Moreover, because of its low toxicity and high biodegradability, SN has negligible effect on cellular function of organs. As the engineered SN can overcome practical challenges associated with therapeutic RNP application, it is strongly expected this platform to be a modular RNPs delivery system, facilitating in vivo gene deletion and editing.

A RUNX1-FPDMM rhesus macaque model reproduces the human phenotype and predicts challenges to curative gene therapies

AbstractGerm line loss-of-function heterozygous mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancies (FPDMM) characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant hematopoietic stem and progenitor cells (HSPCs) in vivo long term. We generated a rhesus macaque with an FPDMM competitive repopulation model using CRISPR/Cas9 nonhomologous end joining editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous HSPCs and tracked mutated allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared with AAVS1-edited cells. Platelet counts remained below the normal range in the long term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous–mutated HSPCs long term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse myelodysplastic syndrome/ acute myeloid leukemia predisposition and thrombopoietic defects.

CRISPR-mediated correction of skeletal muscle Ca2+ handling in a novel DMD patient-derived pluripotent stem cell model

Mutations in the dystrophin gene cause the most common and currently incurable Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting. Although abnormal Ca2+ handling is a pathological feature of DMD, mechanisms underlying defective Ca2+ homeostasis remain unclear. Here we generate a novel DMD patient-derived pluripotent stem cell (PSC) model of skeletal muscle with an isogenic control using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated precise gene correction. Transcriptome analysis identifies dysregulated gene sets in the absence of dystrophin, including genes involved in Ca2+ handling, excitation-contraction coupling and muscle contraction. Specifically, analysis of intracellular Ca2+ transients and mathematical modeling of Ca2+ dynamics reveal significantly reduced cytosolic Ca2+ clearance rates in DMD-PSC derived myotubes. Pharmacological assays demonstrate Ca2+ flux in myotubes is determined by both intracellular and extracellular sources. DMD-PSC derived myotubes display significantly reduced velocity of contractility. Compared with a non-isogenic wildtype PSC line, these pathophysiological defects could be rescued by CRISPR-mediated precise gene correction. Our study provides new insights into abnormal Ca2+ homeostasis in DMD and suggests that Ca2+ signaling pathways amenable to pharmacological modulation are potential therapeutic targets. Importantly, we have established a human physiology-relevant in vitro model enabling rapid pre-clinical testing of potential therapies for DMD.

P.172 AAV-mediated strategy for TCAP gene correction as a new treatment for LGMDR7/LGMD2G dystrophy

P.172 AAV-mediated strategy for TCAP gene correction as a new treatment for LGMDR7/LGMD2G dystrophy

A multifunctional non-viral vector for the delivery of MTH1-targeted CRISPR/Cas9 system for non-small cell lung cancer therapy

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system adapted from bacteria is a programmable nuclease-based genome editing tool. The long-lasting effect of gene silencing or correction is beneficial in cancer treatment. Considering the need to broaden the practical application of this technology, highly efficient non-viral vectors are urgently required. We prepared a multifunctional non-viral vector that could actively target tumor cells and deliver CRISPR/Cas9 plasmids into nuclei of cancer cells. Protamine sulfate (PS) which contains nuclear localization sequence was utilized to condense plasmid DNA and facilitate nuclei-targeted delivery. Liposome-coated protein/DNA complex avoided the degradation of nuclease in blood circulation. The obtained PS@Lip/pCas9 was further modified with distearoyl phosphoethanolamine-polyethylene glycol-hyaluronic acid (HA) to endow the vector ability to actively target tumor cell. Results suggested that PS@HA-Lip could deliver CRISPR/Cas9 plasmids into nuclei of tumor cells and induce genome editing effect. With the disruption of MTH1 (mutT homolog1) gene, the growth of non-small cell lung cancer was inhibited. Moreover, cell apoptosis in tumor tissue was promoted, and liver metastasis of non-small cell lung cancer (NSCLC) was reduced. Our study has provided a therapeutic strategy targeting MTH1 gene for NSCLC therapy. Statement of significanceCRISPR/Cas9 as a powerful tool for genome editing has drawn much attention. The long-lasting effect possesses unique advantage in cancer treatment. Non-viral vectors have high loading capacity, high safety and low immunogenicity, playing an important role in CRISPR/Cas9 delivery. In our study, a multifunctional non-viral vector for the efficient delivery of CRISPR/Cas9 plasmid was constructed. With the active targeting ligand and nuclei-targeting component, the cargo was efficiently delivered into cell nuclei and exerted genome editing effect. By using this vector, we successfully inhibited the growth and induced the apoptosis of non-small cell lung cancer by disrupting MTH1 expression with good safety. Our work provided an efficient non-vial vector for CRISPR/Cas9 delivery and explored the possibility for cancer treatment.