The skin is the largest organ of the body and forms and serves as the barrier for preventing external material from accessing and damaging internal organs. As the outward interface to the environment, it is accessible for the application of therapeutic agents and cellular and gene therapy represent attractive and promising options to treat severe genetic conditions for which palliation has long been the main stay. However, because of its barrier function, transit across and to the subdermal compartment can be challenging. This commentary examines the current approaches of cell and gene therapies for genetic skin disorders. We write this from a local and systemic “outside and inside.” perspective. Delivery from the outside encompasses topical, intradermal, and transdermal strategies for cell and vector delivery and ex vivo cell expansion and grafting. The inside approach details systemic delivery via infusion of cells or agents toward providing benefit to the skin. We use recessive dystrophic epidermolysis bullosa (RDEB) as a representative and paradigmatic disease to showcase these approaches as a means to highlight potential broader applicability to other conditions.

Programmable gene integration in human cells has the potential to enable mutation-agnostic treatments for loss-of-function genetic diseases and facilitate many applications in the life sciences. CRISPR-associated transposases (CASTs) catalyze RNA-guided DNA integration but thus far demonstrate minimal activity in human cells. Using phage-assisted continuous evolution (PACE), we generated CAST variants with >200-fold average improved integration activity. The evolved CAST system (evoCAST) achieves ~10 to 30% integration efficiencies of kilobase-size DNA cargoes in human cells across 14 tested genomic target sites, including safe harbor loci, sites used for immunotherapy, and genes implicated in loss-of-function diseases, with undetected indels and low levels of off-target integration. Collectively, our findings establish a platform for the laboratory evolution of CASTs and advance a versatile system for programmable gene integration in living systems.

This paper presents an electrochemical DNA sensor integrating CRISPR-Cas9 target recognition, gold nanoparticle signal amplification, and a glassy carbon microelectrode array in a four-electrode anodic stripping voltammetry system. The use of microfluidics and a dedicated oxidation electrode enhances gold nanoparticle signal capture and mass transfer efficiency. Applied to lateral flow assay samples, the sensor achieved a detection limit of 5x10(7) copies/mu L, an improvement of two orders of magnitude compared to visual LFA intepretation, enabling more accurate detection of SARS CoV2 nucleic acid amplified by loop-mediated isothermal amplification. This platform significantly advances nucleic acid diagnostics by offering high sensitivity, quantification capability, and compatibility for point-of-collection testing.

Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 and eeBxb1) mediated up to 60% donor integration (3.2-fold that of wild-type Bxb1) in human cell lines with pre-installed recombinase landing sites. In single-transfection experiments at safe-harbour and therapeutically relevant sites, PASSIGE with eeBxb1 led to an average targeted-gene-integration efficiencies of 23% (4.2-fold that of wild-type Bxb1). Notably, integration efficiencies exceeded 30% at multiple sites in primary human fibroblasts. PASSIGE with evoBxb1 or eeBxb1 outperformed PASTE (for ‘programmable addition via site-specific targeting elements’, a method that uses prime editors fused to recombinases) on average by 9.1-fold and 16-fold, respectively. PASSIGE with continuously evolved recombinases is an unusually efficient method for the targeted integration of genes in mammalian cells. Recombinases generated by phage-assisted evolution enhance the efficiency of the prime-editing-assisted targeted integration of large genes in mammalian cells.

Gene editing nucleases, base editors, and prime editors are potential locus-specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa; however, many recessive dystrophic epidermolysis bullosa COL7A1 pathogenic nucleotide variations (PNVs) are unique, making the development of personalized editing reagents challenging. A total of 270 of the ∼320 COL7A1 epidermolysis bullosa PNVs reside in exons that can be skipped, and antisense oligonucleotides and gene editing nucleases have been used to create in-frame deletions. Antisense oligonucleotides are transient, and nucleases generate deleterious double-stranded DNA breaks and uncontrolled mixtures of allele products. We developed a twin prime editing strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon 5. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in recessive dystrophic epidermolysis bullosa fibroblasts, keratinocytes, and induced pluripotent stem cells with minimal double-stranded DNA breaks, and collagen type VII protein was restored. Twin prime editing can replace the target exon with recombinase attachment sequences, and we exploited this to reinsert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twin prime editing can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few double-stranded DNA breaks as a strategy that may enable a single therapeutic agent to treat multiple recessive dystrophic epidermolysis bullosa patient cohorts.

Fanconi anemia (FA) is an inherited DNA repair disorder characterized by bone marrow (BM) failure, developmental abnormalities, myelodysplasia, leukemia, and solid tumor predisposition. Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a mainstay treatment, is limited by conditioning regimen-related toxicity and graft-versus-host disease (GVHD). Antibody-drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) can open marrow niches permitting donor stem cell alloengraftment. Here, we report that single dose anti-mouse CD45-targeted ADC (CD45-ADC) facilitated stable, multilineage chimerism in 3 distinct FA mouse models representing 90% of FA complementation groups. CD45-ADC profoundly depleted host stem cell enriched Lineage(-)Sca1(+)cKit(+) cells within 48 hours. Fanca(-/-) recipients of minor-mismatched BM and single dose CD45-ADC had peripheral blood (PB) mean donor chimerism >90%; donor HSCs alloengraftment was verified in secondary recipients. In Fancc(-/-) and Fancg(-/-) recipients of fully allogeneic grafts, PB mean donor chimerism was 60% to 80% and 70% to 80%, respectively. The mean percent donor chimerism in BM and spleen mirrored PB results. CD45-ADC-conditioned mice did not have clinical toxicity. A transient <2.5-fold increase in hepatocellular enzymes and mild-to-moderate histopathological changes were seen. Under GVHD allo-HSCT conditions, wild-type and Fanca(-/-) recipients of CD45-ADC had markedly reduced GVHD lethality compared with lethal irradiation. Moreover, single dose anti-human CD45-ADC given to rhesus macaque nonhuman primates on days -6 or -10 was at least as myeloablative as lethal irradiation. These data suggest that CD45-ADC can potently promote donor alloengraftment and hematopoiesis without significant toxicity or severe GVHD, as seen with lethal irradiation, providing strong support for clinical trial considerations in highly vulnerable patients with FA.

This paper presents an innovative approach by integrating laser-induced graphene (LIG) as a low-power heater and a CRISPR-Cas9 sensor for efficient DNA amplification and detection. LIG, engineered using a laser-induced carbonization technique on polyimide tape, serves as a rapid heater for isothermal DNA amplification, achieving results within 30 minutes with minimal power consumption. Simultaneously, a CRISPR-Cas9 sensor detects DNA concentrations as low as 16 pM, offering cost-effective and sensitive detection. The combination of LIG heaters and CRISPR-Cas9 sensors streamlines DNA amplification and detection processes, offering substantial advantages over traditional methods in terms of power efficiency, cost effectiveness, and application simplicity. We validated our approach using lateral flow assay technology. Further, a pyrolytic glassy carbon electrode-based electrochemical sensor demonstrates specificity in DNA detection, with a detection limit of 16 pM. This integrated approach holds promise for multiple applications, particularly in nucleic acid diagnostics, showcasing a resource-efficient strategy for DNA amplification and detection in molecular biology research.

Damage to the gastrointestinal tract following allogeneic hematopoietic stem cell transplantation is a significant contributor to the severity and perpetuation of graft-versus-host disease. In preclinical models and clinical trials, we showed that infusing high numbers of regulatory T cells reduces graft-versus-host disease incidence. Despite no change in in vitro suppressive function, transfer of ex vivo expanded regulatory T cells transduced to overexpress G protein-coupled receptor 15 or C-C motif chemokine receptor 9, specific homing receptors for colon or small intestine, respectively, lessened graft-versus-host disease severity in mice. Increased regulatory T cell frequency and retention within the gastrointestinal tissues of mice that received gut homing T cells correlated with lower inflammation and gut damage early post-transplant, decreased graft-versus-host disease severity, and prolonged survival compared with those receiving control transduced regulatory T cells. These data provide evidence that enforced targeting of ex vivo expanded regulatory T cells to the gastrointestinal tract diminishes gut injury and is associated with decreased graft-versus-host disease severity.

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax. We discovered that different reverse transcriptases specialize in different types of edits and used this insight to generate reverse transcriptases that outperform PEmax and PEmaxDRNaseH, the truncated editor used in dualAAV delivery systems. Finally, we generated Cas9 domains that improve prime editing. These resulting editors (PE6a-g) enhance therapeutically relevant editing in patient-derived fibroblasts and primary human T-cells. PE6 variants also enable longer insertions to be installed in vivo following dual-AAV delivery, achieving 40% loxP insertion in the cortex of the murine brain, a 24-fold improvement compared to previous state-of-the-art prime editors.

Wastewater-based epidemiology provides an approach for assessing the prevalence of pathogens such as COVID-19 in a sewer service area. In this study, SARS-CoV-2 RNA was measured serially in 44 wastewater treatment plants of varying service capacities comprising approximately 67% of the population of Minnesota, from September 2020 through December 2022. We employed linear regression models to establish a predictive relationship between the weekly SARS-CoV-2 RNA concentrations in wastewater and clinical case counts. Metrics were assessed under specified transformation and normalization methods which we confirmed by cross-validation averaged across the enrolled treatment plants. We report that the relationship between COVID-19 incidence and SARS-CoV-2 RNA in wastewater may be treatment plant-specific. Toward establishing guidelines for pathogen surveillance, we further studied storage and time-to-analysis for RNA wastewater data and observed large effects of storage temperature, indicating that collection methods may have an important effect on the utility and validity of wastewater data for infectious disease monitoring. Our findings are additive for any large-scale wastewater surveillance program.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe mucocutaneous disease caused by loss-of-function mutations to the collagen VIIa (COL7A1) gene that encodes the type VII collagen peptide (C7). Previously, we used programmable nucleases and DNA base editors (BE) to repair COL7A1 mutations through homology-directed repair (HDR) or deamination, respectively. These efforts represent a personalized medicine approach; however, defined reagents with applicability to multiple patients is desirable. Work has shown that COL7A1 exons with mutations can be excised to create in frame deletions leading to restored C7 production. Nucleases that generate double-stranded DNA breaks (DSBs) have been used to accomplish this; however, DSBs are toxic to the cell and repaired in a stochastic manner that can limit efficiency. Prime editing (PE) is a Cas9-based platform with an associated fused reverse transcriptase that primes off a user provided prime editor guide (peg) RNA that can accommodate any base substitution and insertions and deletions. Twin prime editing (tPE) employs two tandem pegRNAs that can serve to remove large sequences without significant occurrence of DSBs. We used tPE to delete a COL7A1 exon containing an inactivating mutation. A single round of gene transfer showed heterozygous gene targeting and gene restoration in primary, patient-derived fibroblasts and induced pluripotent stem cells (iPSC). Sequencing showed a loss of the target exon with vanishingly small rates of DSB. Corrected fibroblasts represent an approach for autologous gene correction and iPSC can be differentiated in vitro into keratinocytes and mesenchymal stroma cells. The diverse constellation of COL7A1 mutations makes individual patient specific reagents impractical and our data show that tPE offers a regional approach to COL7A1 correction where a small, well characterized set of reagents could be employed across numerous RDEB patients to restore C7 expression by DSB-free in frame pathogenic exon excision.

Recombinant engineering for protein production commonly employs plasmid-based gene templates for introduction and expression of genes in a candidate cell system in vitro. Challenges to this approach include identifying cell types that can facilitate proper post-translational modifications and difficulty expressing large multimeric proteins. We hypothesized that integration of the CRISPR/Cas9-synergistic activator mediator (SAM) system into the human genome would be a powerful tool capable of robust gene expression and protein production. SAMs are comprised of a "dead" Cas9 (dCas9) linked to transcriptional activators viral particle 64 (VP64), nuclear factor-kappa-B p65 subunit (p65), and heat shock factor 1 (HSF1) and are programmable to single or multiple gene targets. We integrated the components of the SAM system into human HEK293, HKB11, SK-HEP1, and HEP-g2 cells using coagulation factor X (FX) and fibrinogen (FBN) as proof of concept. We observed upregulation of mRNA in each cell type with concomitant protein expression. Our findings demonstrate the capability of human cells stably expressing SAM for user-defined singleplex and multiplex gene targeting and highlight their broad potential utility for recombinant engineering as well as transcriptional modulation across networks for basic, translational, and clinical modeling and applications.

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using ‘digital’ genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy.

Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.

Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4(+) T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary human CD4(+) T cells. Using cytosine base editors we observed premature stop codon introduction in up to 89% of sequenced CCR5 or CXCR4 alleles. Using adenine base editors we eliminated the start codon in CCR5 in up to 95% of primary human CD4(+) T cell and up to 88% of CD34(+) hematopoietic stem and progenitor cell target alleles. Genome-wide specificity analysis revealed low numbers of off-target mutations that were introduced by base editing, located predominantly in intergenic or intronic regions. We show that our editing strategies prevent transduction with CCR5-tropic and CXCR4-tropic viral vectors in up to 79% and 88% of human CD4(+) T cells, respectively. The engineered T cells maintained functionality and overall our results demonstrate the effectiveness of base-editing strategies for efficient and specific ablation of HIV co-receptors in clinically relevant cell types.

Current Food and Drug Administration (FDA)-approved CD19-specific chimeric antigen receptor (CAR) T-cell therapies for B-cell malignancies are constitutively active and while efficacious, can cause morbidity and mortality. Their toxicities might be reduced if CAR T-cell activity was regulatable rather than constitutive. To test this, we compared the efficacies and morbidities of constitutively active (conventional) and regulatable (switchable) CAR (sCAR) T-cells specific for human CD19 (huCD19) in an immune-competent huCD19 + transgenic mouse model. Conventional CAR (CAR19) and sCAR T-cells were generated by retrovirally transducing C57BL/6 (B6) congenic T-cells with constructs encoding antibody-derived single chain Fv (sFv) fragments specific for huCD19 or a peptide neoepitope (PNE), respectively. Transduced T-cells were adoptively transferred into huCD19 transgenic hemizygous ( huCD19 Tg/0 ) B6 mice; healthy B-cells in these mice expressed huCD19 Tg . Prior to transfer, recipients were treated with a lymphodepleting dose of cyclophosphamide to enhance T-cell engraftment. In tumor therapy experiments, CAR19 or sCAR T-cells were adoptively transferred into huCD19 Tg/0 mice bearing a syngeneic B-cell lymphoma engineered to express huCD19. To regulate sCAR T cell function, a switch protein was generated that contained the sCAR-specific PNE genetically fused to an anti-huCD19 Fab fragment. Recipients of sCAR T-cells were injected with the switch to link sCAR effector with huCD19 + target cells. Mice were monitored for survival, tumor burden (where appropriate), morbidity (as measured by weight loss and clinical scores), and peripheral blood lymphocyte frequency. CAR19 and sCAR T-cells functioned comparably regarding in vivo expansion and B-cell depletion. However, sCAR T-cells were better tolerated as evidenced by the recipients’ enhanced survival, reduced weight loss, and improved clinical scores. Discontinuing switch administration allowed healthy B-cell frequencies to return to pretreatment levels. In our mouse model, sCAR T-cells killed huCD19 + healthy and malignant B-cells and were better tolerated than CAR19 cells. Our data suggest sCAR might be clinically superior to the current FDA-approved therapies for B-cell lymphomas due to the reduced acute and chronic morbidities and mortality, lower incidence and severity of side effects, and B-cell reconstitution on cessation of switch administration.