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Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of AAV variants is far from being understood, except that genetic recombination plays an important role. Furthermore, little is known about the interaction between AAV and its natural hosts, human and nonhuman primates. In this study, natural AAV capsid genes were subjected to systemic evolutionary analysis with a focus on selection drives during the diversification of AAV lineages. A number of positively selected sites were identified from these AAV lineages with functional relevance implied by their localization on the AAV structures. The selection drives of the two AAV2 capsid sites were further investigated in a series of biological experiments. These observations did not support the evolution of the site 410 of the AAV2 capsid driven by selection pressure from the human CD4+ T-cell response. However, positive selection on site 548 of the AAV2 capsid was directly related to host humoral immunity because of the profound effects of mutations at this site on the immune evasion of AAV variants from human neutralizing antibodies at both the individual and population levels. Overall, this work provides a novel interpretation of the genetic diversity and evolution of AAV lineages in their natural hosts, which may contribute to their further engineering and application in human gene therapy. Author summary: Adeno-associated virus (AAV) is a viral vector that is universally applied in human gene therapy. Despite their versatile applications in gene therapy, the origin and evolution of AAV serotypes have not been extensively studied. In this study, positive selection upon diversification of AAV capsid genes was carefully investigated by evolutionary analysis. Numerous diversifying selected sites were dispersed among the AAV lineages, and evolutionary pathways were inferred for two selected sites of the AAV2 capsid gene. Furthermore, the putative immunological drives underlying the selection of AAV2 capsid genes were experimentally dissected. This work provides a novel interpretation of the evolution and diversity of AAV serotypes and might be helpful for their engineering and application in gene therapy. [ABSTRACT FROM AUTHOR]

The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5′-TTTV-3′ (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research. Cas12a nuclease is commonly used in genome editing applications, but it has been difficult to adopt it for AAV-based gene therapy due to its large size. This study characterizes a novel compact Cas12a ortholog from an uncultured bacteria of the class Erysipelotrichia (EbCas12a) and develops an all-in-one AAV delivery system whereby this EbCas12a can be packaged with its crRNA into a single AAV vector. [ABSTRACT FROM AUTHOR]

Using the CRISPR-Cas9 system to perform base substitutions at the target site is a typical technique for genome editing with the potential for applications in gene therapy and agricultural productivity. When the CRISPR-Cas9 system uses guide RNA to direct the Cas9 endonuclease to the target site, it may misdirect it to a potential off-target site, resulting in an unintended genome editing. Although several computational methods have been proposed to predict off-target effects, there is still room for improvement in the off-target effect prediction capability. In this paper, we present an effective approach called CRISPR-M with a new encoding scheme and a novel multi-view deep learning model to predict the sgRNA off-target effects for target sites containing indels and mismatches. CRISPR-M takes advantage of convolutional neural networks and bidirectional long short-term memory recurrent neural networks to construct a three-branch network towards multi-views. Compared with existing methods, CRISPR-M demonstrates significant performance advantages running on real-world datasets. Furthermore, experimental analysis of CRISPR-M under multiple metrics reveals its capability to extract features and validates its superiority on sgRNA off-target effect predictions. Author summary: Genome editing using the CRISPR-Cas9 system, particularly base substitutions directed by guide RNA, holds immense potential for applications in gene therapy and agricultural productivity. However, the risk of unintended off-target effects poses a challenge, as misdirection of the Cas9 endonuclease can lead to unintended genome alterations. While computational methods exist for predicting off-target effects, there remains a need for encoding methods with more representation space and deep learning models with generalization capability and the adaptability. This paper introduces CRISPR-M, an innovative approach addressing the limitations of existing methods in predicting off-target effects, especially for target sites with indels and mismatches. CRISPR-M employs a novel encoding scheme and a multi-view deep learning model, combining convolutional neural networks and bidirectional long short-term memory recurrent neural networks. The three-branch network structure enhances the prediction accuracy by considering multiple perspectives. Compared with previous representative methods, CRISPR-M exhibits remarkable performance advantages when applied to real-world datasets. The experimental evaluation of CRISPR-M, assessed by various metrics such as ROC, PRC, GC content and melting temperature, demonstrates its ability to extract meaningful features and establishes its superiority in predicting off-target effects of sgRNA. [ABSTRACT FROM AUTHOR]

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus (rAAV) vectors have become a reliable strategy for delivering gene therapies. As rAAV capsid content is known to be heterogeneous, methods for rAAV characterization are critical for assessing the efficacy and safety of drug products. Multiplex digital PCR (dPCR) has emerged as a popular molecular approach for characterizing capsid content due to its high level of throughput, accuracy, and replicability. Despite growing popularity, tools to accurately analyze multiplexed data are scarce. Here, we introduce a novel statistical model to estimate genome integrity from duplex dPCR assays. This work demonstrates that use of a Poisson-multinomial mixture distribution significantly improves the accuracy and quantifiable range of duplex dPCR assays over currently available models. [ABSTRACT FROM AUTHOR]

In a phase I dose escalation and safety study (NCT02555397), a replication-competent oncolytic adenovirus expressing yCD, TK and hIL-12 (Ad5-yCD/mutTKSR39rep-hIL-12) was administered in 15 subjects with localized recurrent prostate cancer (T1c-T2) at increasing doses (1 × 1010, to 1 × 1012 viral particles) followed by 7-day treatment of 5-fluorocytosine (5-FC) and valganciclovir (vGCV). The primary endpoint was toxicity through day 30 while the secondary and exploratory endpoints were quantitation of IL-12, IFNγ, CXCL10 and peripheral blood mononuclear cells (PBMC). The study maximum tolerated dose (MTD) was not reached indicating 1012 viral particles was safe. Total 115 adverse events were observed, most of which (92%) were grade 1/2 that did not require any treatment. Adenoviral DNA was detected only in two patients. Increase in IL-12, IFNγ, and CXCL10 was observed in 57%, 93%, and 79% patients, respectively. Serum cytokines demonstrated viral dose dependency, especially apparent in the highest-dose cohorts. PBMC analysis revealed immune system activation after gene therapy in cohort 5. The PSA doubling time (PSADT) pre and post treatment has a median of 1.55 years vs 1.18 years. This trial confirmed that replication-competent Ad5-IL-12 adenovirus (Ad5-yCD/mutTKSR39rep-hIL-12) was well tolerated when administered locally to prostate tumors. [ABSTRACT FROM AUTHOR]

We report three novel deletions involving the Multispecies Conserved Sequences (MCS) R2, also known as the Major Regulative Element (MRE), in patients showing the α-thalassemia phenotype. The three new rearrangements showed peculiar positions of the breakpoints. 1) The (αα)ES is a telomeric 110 kb deletion ending inside the MCS-R3 element. 2) The (αα)FG, 984 bp-long, ends 51 bp upstream to MCS-R2; both are associated with a severe α-thalassemia phenotype. 3) The (αα)CT, 5058 bp-long starts at position +93 of MCS-R2 and is the only one associated to a mild α-thalassemia phenotype. To understand the specific role of different segments of the MCS-R2 element and of its boundary regions we carried out transcriptional and expression analysis. Transcriptional analysis of patients' reticulocytes showed that (αα)ES was unable to produce α2-globin mRNA, while a high level of expression of the α2-globin genes (56%) was detected in (αα)CT deletion, characterized by the presence of the first 93 bp of MCS-R2. Expression analysis of constructs containing breakpoints and boundary regions of the deletions (αα)CT and (αα)FG, showed comparable activity both for MCS-R2 and the boundary region (-682/-8). Considering that the (αα)CT deletion, almost entirely removing MCS-R2, has a less severe phenotype than the (αα)FG α0thalassemia deletion, removing both MCS-R2 almost entirely and an upstream 679 bp, we infer for the first time that an enhancer element must exist in this region that helps to increase the expression of the α-globin genes. The genotype-phenotype relationship of other previously published MCS-R2 deletions strengthened our hypothesis. Author summary: The regulation of gene expression occurs through the physical interaction between the promoters, regions preceding the genes, and the enhancer, short regions placed even at a considerable distance, with a mechanism defined as long-range regulation. Globin genes are a good model of gene regulation, due to the easy access to the target tissue (blood) and to the analyses that can be performed on it. In a study of patients with alpha-thalassemia, we identified three new deletions involving different parts of the enhancer named Major Regulative Element MCS-R2 associated with severe phenotype, and one of them, with mild. These deletions have given us the opportunity to gain insight into long-range regulation in vivo, using transcriptional analysis on reticulocytes from patients to analyze the role of individual regulatory elements. Our results highlight for the first time a functional enhancer role associated to the boundary MCS-R2 regions and shed light on aspects related to long-range regulation. The data could be useful to better understand the pathophysiology of deletions involving the enhancer MCS-R2 and also for constructing expression vectors for gene therapy and for identifying drug target sequences to modulate gene expression. [ABSTRACT FROM AUTHOR]

With the advent of recombinant DNA technology in the 1970s, the idea of using gene therapies to treat human genetic diseases captured the interest and imagination of scientists around the world. Years later, enabled largely by the development of CRISPR-based genome editing tools, the field has exploded, with academic labs, startup biotechnology companies, and large pharmaceutical corporations working in concert to develop life-changing therapeutics. In this Essay, we highlight base editing technologies and their development from bench to bedside. Base editing, first reported in 2016, is capable of installing C•G to T•A and A•T to G•C point mutations, while largely circumventing some of the pitfalls of traditional CRISPR/Cas9 gene editing. Despite their youth, these technologies have been widely used by both academic labs and therapeutics-based companies. Here, we provide an overview of the mechanics of base editing and its use in clinical trials. Base editing, first reported in 2016, is a genome editing technology capable of installing point mutations with high efficiency and precision. This Essay discusses base editing technologies and their development from bench to bedside. [ABSTRACT FROM AUTHOR]

Precise, reproducible characterization of AAV is critical for comparing preclinical results between laboratories and determining a safe and effective clinical dose for gene therapy applications. In this study, we systematically evaluated numerous parameters to produce a simple and robust ddPCR protocol for AAV characterization. The protocol uses a low ionic strength buffer containing Pluronic-F68 and polyadenylic acid to dilute the AAV into the ddPCR concentration range and a 10-minute thermal capsid lysis prior to assembling ddPCR reactions containing MspI. A critical finding is that the buffer composition affected the ITR concentration of AAV but not the ITR concentration of a double stranded plasmid, which has implications when using a theoretical, stoichiometric conversion factor to obtain the titer based on the ITR concentration. Using this protocol, a more comprehensive analysis of an AAV vector formulation was demonstrated with multiple ddPCR assays distributed throughout the AAV vector genome. These assays amplify the ITR, regulatory elements, and eGFP transgene to provide a more confident estimate of the vector genome concentration and a high-resolution characterization of the vector genome identity. Additionally, we compared two methods of genome integrity analysis for three control sample types at eight different concentrations for each sample. The genome integrity was independent of sample concentration and the expected values were obtained when integrity was determined based on the excess number of positive droplets relative to the number of double positive droplets expected by chance co-encapsulation of two DNA targets. The genome integrity was highly variable and produced unexpected values when the double positive droplet percentage was used to calculate the genome integrity. A protocol using a one-minute thermal capsid lysis prior to assembling ddPCR reactions lacking a restriction enzyme used the non-ITR assays in a duplex ddPCR milepost experiment to determine the genome integrity using linkage analysis. [ABSTRACT FROM AUTHOR]

Targeting transgene integration into a safe genomic locus would be very important for gene therapy. We have generated lentivirus vectors containing the ribosomal RNA-recognising I-PpoI endonuclease fused to viral integrase, and transgene cassettes with target site homology arms to enhance insertion targeting. These new vectors were characterised with respect to the persistence of transgene expression, insertion targeting efficiency and chromosomal integrity of the transduced cells. The aim was to find an optimally safe and effective vector for human gene therapy. Fusion protein vectors with high endonuclease activity were the most effective in the accurate targeting of transgene insertion. The homology construct increased the insertion targeting efficiency to 28% in MRC-5 cells. However, karyotyping analysis showed that the high endonuclease activity induced the formation of derivative chromosomes in as many as 24% of the analysed primary T lymphocytes. The persistence of transgene expression was excellent in homology arm-containing fusion protein vectors with reduced endonuclease activity, and these fusion proteins did not cause any detectable chromosomal rearrangements attributable to the endonuclease activity. We thus conclude that instead of the fusion protein vectors that carry a highly active endonuclease, our vectors with the ability to tether the lentivirus preintegration complex to benign loci in the genome without high ribosomal DNA cleavage activity are better suited for lentivirus-based gene therapy applications. [ABSTRACT FROM AUTHOR]

The small size of CjCas9 can make easier its vectorization for in vivo gene therapy. However, compared to the SpCas9, the CjCas9 is, in general, less efficient to generate indels in target genes. The factors that affect its efficacity are not yet determined. We observed that the CjCas9 protein expressed in HEK293T cells after transfection of this transgene under a CMV promoter was much lower than the SpCas9 protein in the same conditions. We thus evaluated the effect of proteasome inhibitors on CjCas9 protein stability and its efficiency on FXN gene editing. Western blotting showed that the addition of MG132 or bortezomib, significantly increased CjCas9 protein levels in HEK293T and HeLa cells. Moreover, bortezomib increased the level of CjCas9 protein expressed under promoters weaker than CMV such as CBH or EFS but which are specific for certain tissues. Finally, ddPCR quantification showed that bortezomib treatment enhanced CjCas9 efficiency to delete GAA repeat region of FXN gene in HEK293T cells. The improvement of CjCas9 protein stability would facilitate its used in CRISPR/Cas system. [ABSTRACT FROM AUTHOR]

Due to its close resemblance, the domesticated pig has proven to be a diverse animal model for biomedical research and genome editing tools have contributed to developing porcine models for several human diseases. By employing the CRISPR-Cas9 system, porcine embryos or somatic cells can be genetically modified to generate the desired genotype. However, somatic cell nuclear transfer (SCNT) of modified somatic cells and embryo manipulation are challenging, especially if the desired genotype is detrimental to the embryo. Direct in vivo edits may facilitate the production of genetically engineered pigs by integrating Cas9 into the porcine genome. Cas9 expressing cells were generated by either random integration or transposon-based integration of Cas9 and used as donor cells in SCNT. In total, 15 animals were generated that carried a transposon-based Cas9 integration and two pigs a randomly integrated Cas9. Cas9 expression was confirmed in muscle, tonsil, spleen, kidney, lymph nodes, oral mucosa, and liver in two boars. Overall, Cas9 expression was higher for transposon-based integration, except in tonsils and liver. To verify Cas9 activity, fibroblasts were subjected to in vitro genome editing. Isolated fibroblasts were transfected with guide RNAs (gRNA) targeting different genes (GGTA1, B4GALNT2, B2M) relevant to xenotransplantation. Next generation sequencing revealed that the editing efficiencies varied (2–60%) between the different target genes. These results show that the integrated Cas9 remained functional, and that Cas9 expressing pigs may be used to induce desired genomic modifications to model human diseases or further evaluate in vivo gene therapy approaches. [ABSTRACT FROM AUTHOR]

Objective: To evaluate efficacy of a novel adeno-associated virus (AAV) vector, AAV2/4-RS1, for retinal rescue in the retinoschisin knockout (Rs1-KO) mouse model of X-linked retinoschisis (XLRS). Brinzolamide (Azopt®), a carbonic anhydrase inhibitor, was tested for its ability to potentiate the effects of AAV2/4-RS1. Methods: AAV2/4-RS1 with a cytomegalovirus (CMV) promoter (2x1012 viral genomes/mL) was delivered to Rs1-KO mice via intravitreal (N = 5; 1μL) or subretinal (N = 21; 2μL) injections at postnatal day 60–90. Eleven mice treated with subretinal therapy also received topical Azopt® twice a day. Serial full field electroretinography (ERG) was performed starting at day 50–60 post-injection. Mice were evaluated using a visually guided swim assay (VGSA) in light and dark conditions. The experimental groups were compared to untreated Rs1-KO (N = 11), wild-type (N = 12), and Rs1-KO mice receiving only Azopt® (N = 5). Immunofluorescence staining was performed to assess RS1 protein expression following treatment. Results: The ERG b/a ratio was significantly higher in the subretinal plus Azopt® (p<0.0001), subretinal without Azopt® (p = 0.0002), and intravitreal (p = 0.01) treated eyes compared to untreated eyes. There was a highly significant subretinal treatment effect on ERG amplitudes collectively at 7–9 months post-injection (p = 0.0003). Cones showed more effect than rods. The subretinal group showed improved time to platform in the dark VGSA compared to untreated mice (p<0.0001). RS1 protein expression was detected in the outer retina in subretinal treated mice and in the inner retina in intravitreal treated mice. Conclusions: AAV2/4-RS1 shows promise for improving retinal phenotype in the Rs1-KO mouse model. Subretinal delivery was superior to intravitreal. Topical brinzolamide did not improve efficacy. AAV2/4-RS1 may be considered as a potential treatment for XLRS patients. [ABSTRACT FROM AUTHOR]

Equine recurrent uveitis (ERU) is a spontaneous, painful, and vision threatening disease affecting up to 25% of equine populations worldwide. Current treatments of ERU are non-specific and have many side effects which limits them to short-term use. In order to develop an effective therapy for ERU, we investigated the use of adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by equine interleukin-10 (Equine-IL10). The purpose of this study was to evaluate the therapeutic efficacy of a single intravitreal (IVT) dose of AAV8-Equine-IL10 gene therapy for inhibition of experimental autoimmune uveitis (EAU) in rats. Each rat was dosed intravitreally (IVT) in both eyes with either balanced salt solution (BSS) (control; n = 4), AAV8-Equine-IL10 at a low dose (2.4x109 vg; n = 5) or high dose (2.4x1010 vg; n = 5). EAU was induced in all groups of rats 7 days after IVT injections and euthanized 21 days post-injection. Ophthalmic examination and aqueous humor (AH) cell counts were recorded with the observer blinded to the treatment groups. Histopathology and qPCR were performed on selected ocular tissues. Data presented herein demonstrate that AAV8-Equine-IL10 treated rats exhibited a significant decrease in clinical inflammatory scores and AH cell counts compared to BSS-treated EAU eyes on days 10, 12 and 14 post EAU induction at both administered vector doses. Mean cellular histologic infiltrative scores were also significantly less in AAV8-Equine-IL10 dosed rats compared to the BSS group. Intravitreal injection of AAV8-Equine-IL10 resulted in Equine-IL10 cDNA expression in the ciliary body, retina, cornea, and optic nerve in a dose-dependent manner. A single IVT injection of AAV8-Equine-IL10 appeared to be well-tolerated and inhibited EAU even at the lowest administered dose. These results demonstrate safety and efficacy of AAV8-Equine-IL10 to prevent EAU and support continued exploration of AAV gene therapy for the treatment of equine and perhaps human recurrent uveitis. [ABSTRACT FROM AUTHOR]

Hearing impairment (HI) is a heterogeneous condition that affects many individuals globally with different age groups. HI is a genetically and phenotypically heterogeneous disorder. Over the last several years, many genes/loci causing rare autosomal recessive and dominant forms of hearing impairments have been identified, involved in various aspects of ear development. In the current study, two affected individuals of a consanguineous family exhibiting autosomal recessive nonsyndromic hearing impairment (AR-NSHI) were clinically and genetically characterized. The single affected individual (IV-2) of the family was subjected to whole-exome sequencing (WES) accompanied by traditional Sanger sequencing. Clinical examinations using air conduction audiograms of both the affected individuals showed profound hearing loss across all frequencies. WES revealed a homozygous missense variant (c.44G>C) in the SIX5 gene located on chromosome 19q13.32. We report the first case of autosomal recessive NSHI due to a biallelic missense variant in the SIX5 gene. This report further supports the evidence that the SIX5 variant might cause profound HI and supports its vital role in auditory function. Identification of novel candidate genes might help in application of future gene therapy strategies that may be implemented for NSHI, such as gene replacement using cDNA, gene silencing using RNA interference, and gene editing using the CRISPR/Cas9 system. [ABSTRACT FROM AUTHOR]

In spite of continuous development of gene therapy vectors with thousands of drug candidates in clinical drug trials there are only a small number approved on the market today stressing the need to have characterization methods to assist in the validation of the drug development process. The level of packaging of the vector capsids appears to play a critical role in immunogenicity, hence an objective quantitative method assessing the content of particles containing a genome is an essential quality measurement. As transmission electron microscopy (TEM) allows direct visualization of the particles present in a specimen, it naturally seems as the most intuitive method of choice for characterizing recombinant adeno-associated virus (rAAV) particle packaging. Negative stain TEM (nsTEM) is an established characterization method for analysing the packaging of viral vectors. It has however shown limitations in terms of reliability. To overcome this drawback, we propose an analytical method based on CryoTEM that unambiguously and robustly determines the percentage of filled particles in an rAAV sample. In addition, we show that at a fixed number of vector particles the portion of filled particles correlates well with the potency of the drug. The method has been validated according to the ICH Q2 (R1) guidelines and the components investigated during the validation are presented in this study. The reliability of nsTEM as a method for the assessment of filled particles is also investigated along with a discussion about the origin of the observed variability of this method. [ABSTRACT FROM AUTHOR]

Many viruses utilize the lymphohematogenous route for dissemination; however, they may not freely use this highway unchecked. The reticuloendothelial system (RES) is an innate defense system that surveys circulating blood, recognizing and capturing viral particles. Examination of the literature shows that the bulk of viral clearance is mediated by the liver; however, the precise mechanism(s) mediating viral vascular clearance vary between viruses and, in many cases, remains poorly defined. Herein, we summarize what is known regarding the recognition and capture of virions from the circulation prior to the generation of a specific antibody response. We also discuss the consequences of viral capture on viral pathogenesis and the fate of the captor cell. Finally, this understudied topic has implications beyond viral pathogenesis, including effects on arbovirus ecology and the application of virus-vectored gene therapies. Author summary: Limiting the amount of virus freely circulating in the bloodstream can be important for controlling viral pathogenesis and transmission. However, despite early advances, this field of study has become overlooked and understudied. Innate immune cells in the liver and spleen constantly survey and remove from circulation viral particles without the aid of virus-specific antibody. The details of these host–virus interactions, and the consequences thereof, remain unknown for many viruses. Yet, understanding this phenomenon has implications not only on bettering our understanding of disease progress, but also on arbovirus ecology and the development of effective virus-vectored gene therapies. [ABSTRACT FROM AUTHOR]

The role played by certain domestic species such as dogs as a translational model in comparative oncology shows great interest to develop new therapeutic strategies in brain tumors. Gliomas are a therapeutic challenge that represents the most common form of malignant primary brain tumors in humans and the second most common form in dogs. Gene-directed enzyme/prodrug therapy using adipose mesenchymal stem cells (Ad-MSCs) expressing the herpes simplex virus thymidine kinase (TK) has proven to be a promising alternative in glioblastoma therapy, through its capacity to migrate and home to the tumor and delivering local cytotoxicity avoiding other systemic administration. In this study, we demonstrate the possibility for canine Ad-MSCs (cAd-MSCs) to be genetically engineered efficiently with a lentiviral vector to express TK (TK-cAd-MSCs) and in combination with ganciclovir (GCV) prodrug demonstrated its potential antitumor efficacy in vitro and in vivo in a mice model with the human glioblastoma cell line U87. TK-cAd-MSCs maintained cell proliferation, karyotype stability, and MSCs phenotype. Genetic modification significantly affects its secretory profile, both the analyzed soluble factors and exosomes. TK-cAd-MSCs showed a high secretory profile of some active antitumor immune response cytokines and a threefold increase in the amount of secreted exosomes, with changes in their protein cargo. We also found that the prodrug protein is not released directly into the culture medium by TK-cAd-MSCs. We believe that our work provides new perspectives for glioblastoma gene therapy in dogs and a better understanding of this therapy in view of its possible implantation in humans. [ABSTRACT FROM AUTHOR]

Adeno-associated viruses (AAV) are considered non-pathogenic in humans, and thus have been developed into powerful vector platforms for in vivo gene therapy. Although the various AAV serotypes display broad tropism, frequently infecting multiple tissues and cell types, vectors for specific and efficient targeting of human CD4+ T lymphocytes are largely missing. In fact, a substantial translational bottleneck exists in the field of therapeutic gene transfer that would require in vivo delivery into peripheral disease-related lymphocytes for subsequent genome editing. To solve this issue, capsid modification for retargeting AAV tropism, and in turn improving vector potency, is considered a promising strategy. Here, we genetically modified the minor AAV2 capsid proteins, VP1 and VP2, with a set of novel nanobodies with high-affinity for the human CD4 receptor. These novel vector variants demonstrated improved targeting of human CD4+ cells, including primary human peripheral blood mononuclear cells (PBMC) and purified human CD4+ T lymphocytes. Thus, the technical approach presented here provides a promising strategy for developing specific gene therapy vectors, particularly targeting disease-related peripheral blood CD4+ leukocytes. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis. Author summary: During the COVID-19 pandemic, variants of SARS-CoV-2 have emerged that are more deadly, more transmissible, and that evade immune response. These variants threaten to reduce the efficacy of first-generation vaccine and monoclonal antibody therapeutics. An additional problem with the existing vaccine strategy is that immune-compromised patients do not respond to active vaccinations. To address these issues, we developed a gene therapy-based strategy to prevent COVID-19 using a viral vector delivered by nasal spray. Unlike vaccines, this passive prophylaxis does not require that the patient mount an immune response. Instead, airway cells are engineered to secrete an antiviral decoy protein that can intercept viral particles before they cause infection. This decoy is based on human ACE2, the natural receptor for SARS-CoV-2. This design imparts resistance to SARS-CoV-2 viral evolution: viral mutations that evade the ACE2 decoy are unlikely to emerge because they would also render the virus less capable of infecting cells and therefore be less fit. Indeed, our ACE2 decoy neutralizes all SARS-CoV-2 variants that we tested and even binds distantly related coronaviruses that have not yet crossed over from animals to humans. This suggests that the ACE2 decoy gene therapy approach could be effectively deployed to vulnerable populations in the COVID-19 pandemic and potentially to broader populations at the outset of future coronavirus pandemics. [ABSTRACT FROM AUTHOR]

Homologous recombination (HR) faithfully restores DNA double-strand breaks. Defects in this HR repair pathway are associated with cancer predisposition. In genetic engineering, HR has been used extensively to study gene function and it represents an ideal method of gene therapy for single gene disorders. Here, we present a novel assay to measure HR in living cells. The HR substrate consisted of a non-fluorescent 3' truncated form of the eGFP gene and was integrated into the AAVS1 locus, known as a safe harbor. The donor DNA template comprised a 5' truncated eGFP copy and was delivered via AAV particles. HR mediated repair restored full-length eGFP coding sequence, resulting in eGFP+ cells. The utility of our assay in quantifying HR events was validated by exploring the impact of the overexpression of HR promoters and the siRNA-mediated silencing of genes known to play a role in DNA repair on the frequency of HR. We conclude that this novel assay represents a useful tool to further investigate the mechanisms that control HR and test continually emerging tools for HR-mediated genome editing. [ABSTRACT FROM AUTHOR]

In contrast to AAV, Simian Virus 40 (rSV40) not inducing neutralizing antibodies (NAbs) allowing re-treatment seems a promising vector for neonatal treatment of inherited liver disorders. Several studies have reported efficacy of rSV40 in animal models for inherited liver diseases. In all studies the ubiquitous endogenous early promoter controlled transgene expression establishing expression in all transduced tissues. Restricting this expression to the target tissues reduces the risk of immune response to the therapeutic gene. In this study a liver specific rSV40 vector was generated by inserting a hepatocyte specific promoter. This increased the specificity of the expression of hUGT1A1 in vitro. However, in vivo the efficacy of rSV40 appeared too low to demonstrate tissue specificity while increasing the vector dose was not possible because of toxicity. In contrast to earlier studies, neutralizing antibodies were induced. Overall, the lack of a platform to produce high titered and pure rSV40 particles and the induction of NAbs, renders it a poor candidate for in vivo gene therapy. [ABSTRACT FROM AUTHOR]

Gene editing has become an essential tool for interrogation of gene function in biomedical research and is also a promising approach for gene therapy. Despite recent progresses, the gene-editing procedure is still a tedious process involving manually isolating large number of single cell colonies to screen for desired mutations. For diploid eukaryotic cells, there is the additional challenge to inactivate both alleles for genes-of-interest, i.e., generating double knockouts (DKOs), for the desired phenotypes or therapeutic effects. In this report, we present a novel method based on Fluorescence Assisted Cell Sorting (FACS) to enrich for DKO cells, using a cell surface marker β2-microglobulin (B2M) as a basis for negative selection. This method significantly increased percentage of DKOs in isolated cells after gene editing, and in the meantime, significantly improve the efficiency of workflow by automating colony isolation. It would greatly facilitate future biomedical research including potential gene/cell therapies. [ABSTRACT FROM AUTHOR]

Objective: Gremlin 1 (GREM1) is a secreted BMP2/4 inhibitor which regulates commitment and differentiation of human adipose precursor cells and prevents the browning effect of BMP4. GREM1 is an insulin antagonist and serum levels are high in type 2 diabetes (T2D). We here examined in vivo effects of AAV8 (Adeno-Associated Viral vectors of serotype eight) GREM 1 targeting the liver in mature mice to increase its systemic secretion and also, in a separate study, injected recombinant GREM 1 intraperitoneally. The objective was to characterize systemic effects of GREM 1 on insulin sensitivity, glucose tolerance, body weight, adipose cell browning and other local tissue effects. Methods: Adult mice were injected with AAV8 vectors expressing GREM1 in the liver or receiving regular intra-peritoneal injections of recombinant GREM1 protein. The mice were fed with a low fat or high fat diet (HFD) and followed over time. Results: Liver-targeted AAV8-GREM1 did not alter body weight, whole-body glucose and insulin tolerance, or adipose tissue gene expression. Although GREM1 protein accumulated in liver cells, GREM1 serum levels were not increased suggesting that it may not have been normally processed for secretion. Hepatic lipid accumulation, inflammation and fibrosis were also not changed. Repeated intraperitoneal rec-GREM1 injections for 5 weeks were also without effects on body weight and insulin sensitivity. UCP1 was slightly but significantly reduced in both white and brown adipose tissue but this was not of sufficient magnitude to alter body weight. We validated that recombinant GREM1 inhibited BMP4-induced pSMAD1/5/9 in murine cells in vitro, but saw no direct inhibitory effect on insulin signalling and pAkt (ser 473 and thr 308) activation. Conclusion: GREM1 accumulates intracellularly when overexpressed in the liver cells of mature mice and is apparently not normally processed/secreted. However, also repeated intraperitoneal injections were without effects on body weight and insulin sensitivity and adipose tissue UCP1 levels were only marginally reduced. These results suggest that mature mice do not readily respond to GREMLIN 1 but treatment of murine cells with GREMLIN 1 protein in vitro validated its inhibitory effect on BMP4 signalling while insulin signalling was not altered. [ABSTRACT FROM AUTHOR]

The cocaine vaccine dAd5GNE is comprised of a disrupted serotype 5 adenovirus gene therapy vector covalently conjugated to the cocaine analog GNE. The vaccine evokes a high titer of circulating anti-cocaine antibodies that prevent cocaine from reaching its cognate receptors in the central nervous system. Prior studies have demonstrated the efficacy of dAd5GNE in models of occasional, moderate cocaine use. However, previous studies have not sufficiently evaluated the efficacy of dAd5GNE in models of the repetitive and high-dose "binge" use patterns common in human addicts. In the present study, we evaluated the capacity of dAd5GNE vaccination to protect against "binge" cocaine use and circumstances where vaccinated addicts attempt to override the vaccine. We modeled repetitive daily cocaine use in vaccinated Balb/c mice and African green monkeys, and evaluated high-dose "binge" scenarios in Balb/c mice. In each model of daily use the dAd5GNE vaccine prevented cocaine from reaching the central nervous system. In the high-dose "binge" model, vaccination decreased cocaine-induced hyperactivity and reduced the number of cocaine-induced seizures. Based on this data and our prior data in rodents and nonhuman primates, we have initiated a clinical trial evaluating the dAd5GNE anti-cocaine vaccine as a potential therapy for cocaine addicts who wish to stop cocaine use. If dAd5GNE vaccination is safe and produces high anti-cocaine antibody titers in the clinic, we hypothesize that the vaccine will restrict the access of cocaine to the central nervous system and inhibit cocaine-induced "highs" even in the context of moderate daily and high-dose "binge" use that might otherwise cause a drug-induced overdose. [ABSTRACT FROM AUTHOR]

Objectives: Colorectal cancer (CRC) is one of the most common malignant human tumors. It is associated with high morbidity and mortality rates. In recent years, tumor gene therapy has emerged as a promising new approach for colorectal cancer therapy. Herein, we identify and analyze the role of COPB2 (coatomer protein complex, subunit beta 2) in proliferation and apoptosis of CRC cells. Methods: To investigate the role of COPB2 in the proliferation and apoptosis of CRC cells, a shCOPB2 vector and a shCtrl vector were constructed for transfection into RKO and HCT116 cells. Cells proliferation was subsequently measured via cell counting kit-8 (CCK8) assay and Celigo cell counting assay. Apoptosis was measured via flow cytometry. The activity level of Caspase 3/7 was measured. Finally, the level of several JNK/c-Jun apoptosis pathway-related proteins were measured to characterize the mechanism of apoptosis. Results: Our results showed that the proliferation rate was decreased and the apoptosis rate was increased in shCOPB2-treated RKO and HCT116 cells compared to those in controls. After the silencing of COPB2, JNK/c-Jun signal pathway activation was increased, the expression levels of apoptosis pathway-related proteins, such as Bad, p53 and Caspase 3, were also increased. Conclusion: COPB2 gene silencing can inhibit RKO and HCT116 cells proliferation and induce apoptosis via the JNK/c-Jun signaling pathway. [ABSTRACT FROM AUTHOR]

Gene therapy is a promising treatment option for cancer. However, its utility may be limited due to expression in off-target cells. Cancer-specific promoters such as telomerase reverse transcriptase (TERT), survivin, and chemokine receptor 4 (CXCR4) have enhanced activity in a variety of human and murine cancers, however, little has been published regarding these promoters in dogs. Given the utility of canine cancer models, the activity of these promoters along with adenoviral E2F enhanced E1a promoter (EEE) was evaluated in a variety of canine tumors, both from the endogenous gene and from exogenously administered constructs. Endogenous expression levels were measured for cTERT, cSurvivin, and cCXCR4 and were low for all three, with some non-malignant and some tumor cell lines and tissues expressing the gene. Expression levels from exogenously supplied promoters were measured by both the number of cells expressing the construct and the intensity of expression in individual cells. Exogenously supplied promoters were active in more cells in all tumor lines than in normal cells, with the EEE promoter being most active, followed by cTERT. The intensity of expression varied more with cell type than with specific promoters. Ultimately, no single promoter was identified that would result in reliable expression, regardless of the tumor type. Thus, these findings imply that identification of a pan-cancer promoter may be difficult. In addition, this data raises the concern that endogenous expression analysis may not accurately predict exogenous promoter activity. [ABSTRACT FROM AUTHOR]

Mammary carcinoma, including triple-negative breast carcinomas (TNBC) are tumor-types for which human and canine pathologies are closely related at the molecular level. The efficacy of an oncolytic vaccinia virus (VV) was compared in low-passage primary carcinoma cells from TNBC versus non-TNBC. Non-TNBC cells were 28 fold more sensitive to VV than TNBC cells in which VV replication is impaired. Single-cell RNA-seq performed on two different TNBC cell samples, infected or not with VV, highlighted three distinct populations: naïve cells, bystander cells, defined as cells exposed to the virus but not infected and infected cells. The transcriptomes of these three populations showed striking variations in the modulation of pathways regulated by cytokines and growth factors. We hypothesized that the pool of genes expressed in the bystander populations was enriched in antiviral genes. Bioinformatic analysis suggested that the reduced activity of the virus was associated with a higher mesenchymal status of the cells. In addition, we demonstrated experimentally that high expression of one gene, DDIT4, is detrimental to VV production. Considering that DDIT4 is associated with a poor prognosis in various cancers including TNBC, our data highlight DDIT4 as a candidate resistance marker for oncolytic poxvirus therapy. This information could be used to design new generations of oncolytic poxviruses. Beyond the field of gene therapy, this study demonstrates that single-cell transcriptomics can be used to identify cellular factors influencing viral replication. Author summary: The identification of cellular genes influencing viral replication/propagation has been studied using hypothesis-driven approaches and/or high-throughput RNA interference screens. In the present report, we propose a methodology based on single-cell transcriptomics. We have studied, in the context of oncolytic virotherapy, the susceptibility of different grades of primary low-passage mammary carcinoma cells of canine origin to an oncolytic vaccinia virus (VV). We highlight a fault in replication of VV in cells that originated from high-grade triple-negative breast carcinomas (TNBC). Single-cell RNA-seq performed on TNBC cell samples infected with VV suggested that the reduced activity of the virus was associated with a higher mesenchymal status of the cells. We also demonstrate that high expression of one gene, DDIT4, is detrimental to VV production. Considering that DDIT4 is associated with a poor prognosis in various cancers including TNBC, our data highlight DDIT4 as a candidate resistance marker for oncolytic poxvirus therapy. Beyond the field of cancer gene therapy, we demonstrate here that single-cell transcriptomics increases the arsenal of tools available to identify cellular factors influencing viral replication. [ABSTRACT FROM AUTHOR]

Wilson's disease (WD) is a monogenetic liver disease that is based on a mutation of the ATP7B gene and leads to a functional deterioration in copper (Cu) excretion in the liver. The excess Cu accumulates in various organs such as the liver and brain. WD patients show clinical heterogeneity, which can range from acute or chronic liver failure to neurological symptoms. The course of the disease can be improved by a life-long treatment with zinc or chelators such as D-penicillamine in a majority of patients, but serious side effects have been observed in a significant portion of patients, e.g. neurological deterioration and nephrotoxicity, so that a liver transplant would be inevitable. An alternative therapy option would be the genetic correction of the ATP7B gene. The novel gene therapy method CRISPR/Cas9, which has recently been used in the clinic, may represent a suitable therapeutic opportunity. In this study, we first initiated an artificial ATP7B point mutation in a human cell line using CRISPR/Cas9 gene editing, and corrected this mutation by the additional use of single-stranded oligo DNA nucleotides (ssODNs), simulating a gene correction of a WD point mutation in vitro. By the addition of 0.5 mM of Cu three days after lipofection, a high yield of CRISPR/Cas9-mediated ATP7B repaired cell clones was achieved (60%). Moreover, the repair efficiency was enhanced using ssODNs that incorporated three blocking mutations. The repaired cell clones showed a high resistance to Cu after exposure to increasing Cu concentrations. Our findings indicate that CRISPR/Cas9-mediated correction of ATP7B point mutations is feasible and may have the potential to be transferred to the clinic. [ABSTRACT FROM AUTHOR]

The EGFR-targeting cancer therapies are commonly facing drug resistance, mostly due to mutations. Gene therapy with artificial microRNA targeting EGFR conserved sequence may avoid such problem. In this study, we constructed a recombinant adenovirus expressing EGFR-targeting artificial microRNA and active revCASP3 (Ad-EC), under the control of tumor-specific SLPI promoter, and evaluated its inhibitory effect on HEP-2 cancer cells both in vitro and in vivo. MTT assay showed that cell growth inhibition rate at 72h was 44.0% in Ad-EC group at MOI 50, while the rate was 7.7% in the control virus Ad-GFP group and 3.6% in Cetuximab (500 μg/ml) group respectively. Flow cytometry analysis revealed the late apoptotic cells rate was 36.1% in Ad-EC group, significantly higher than 6.5% of Ad-GFP group (p < 0.001). When Ad-EC (MOI 50) was combined with CDDP (0.25 μg/ml), late apoptotic cells rate increased to 61.2%, significantly higher than each monotherapy group (P < 0.001). The real-time xCELLigence system recorded an effective cell growth inhibition in Ad-EC and CDDP groups, and more enhanced effect in Ad-EC plus CDDP group. Western blot revealed that Ad-EC could inhibit the activation of AKT pathway and ERK1/2 pathway, while Cetuximab had the AKT pathway over-activated. In vivo experiments with HEP-2 xenograft in nude mice confirmed the tumor inhibition in Ad-EC, CDDP and Ad-EC plus CDDP groups compared with PBS group (P < 0.01). Collectively, these data support the effective inhibition of cancer cells by this novel gene therapy strategy. [ABSTRACT FROM AUTHOR]

Compact CRISPR/Cas9 systems that can be packaged into an adeno-associated virus (AAV) hold great promise for gene therapy. Unfortunately, currently available small Cas9 nucleases either display low activity or require a long protospacer adjacent motif (PAM) sequence, limiting their extensive applications. Here, we screened a panel of Cas9 nucleases and identified a small Cas9 ortholog from Staphylococcus auricularis (SauriCas9), which recognizes a simple NNGG PAM, displays high activity for genome editing, and is compact enough to be packaged into an AAV for genome editing. Moreover, the conversion of adenine and cytosine bases can be achieved by fusing SauriCas9 to the cytidine and adenine deaminase. Therefore, SauriCas9 holds great potential for both basic research and clinical applications. A small ortholog of Cas9 isolated from Staphylococcus auricularis (SauriCas9), which recognizes a simple 5'-NNGG-3' protospacer adjacent motif (PAM), displays high activity for genome editing and is compact enough to be packaged into adeno-associated virus for genome editing. [ABSTRACT FROM AUTHOR]

Background: Validation of previously identified candidate biomarkers and identification of additional candidate gene expression profiles to facilitate diagnosis of tuberculosis (TB) disease and monitoring treatment responses in the Ethiopian context is vital for improving TB control in the future. Methods: Expression levels of 105 immune-related genes were determined in the blood of 80 HIV-negative study participants composed of 40 active TB cases, 20 latent TB infected individuals with positive tuberculin skin test (TST+), and 20 healthy controls with no Mycobacterium tuberculosis (Mtb) infection (TST-), using focused gene expression profiling by dual-color Reverse-Transcription Multiplex Ligation-dependent Probe Amplification assay. Gene expression levels were also measured six months after anti-TB treatment (ATT) and follow-up in 38 TB patients. Results: The expression of 15 host genes in TB patients could accurately discriminate between TB cases versus both TST+ and TST- controls at baseline and thus holds promise as biomarker signature to classify active TB disease versus latent TB infection in an Ethiopian setting. Interestingly, the expression levels of most genes that markedly discriminated between TB cases versus TST+ or TST- controls did not normalize following completion of ATT therapy at 6 months (except for PTPRCv1, FCGR1A, GZMB, CASP8 and GNLY) but had only fully normalized at the 18 months follow-up time point. Of note, network analysis comparing TB-associated host genes identified in the current HIV-negative TB cohort to TB-associated genes identified in our previously published Ethiopian HIV-positive TB cohort, revealed an over-representation of pattern recognition receptors including TLR2 and TLR4 in the HIV-positive cohort which was not seen in the HIV-negative cohort. Moreover, using ROC cutoff ≥ 0.80, FCGR1A was the only marker with classifying potential between TB infection and TB disease regardless of HIV status. Conclusions: Our data indicate that complex gene expression signatures are required to measure blood transcriptomic responses during and after successful ATT to fully diagnose TB disease and characterise drug-induced relapse-free cure, combining genes which resolve completely during the 6-months treatment phase of therapy with genes that only fully return to normal levels during the post-treatment resolution phase. [ABSTRACT FROM AUTHOR]

Rare diseases defined by genetic mutations are classic targets for gene therapy. More recently, researchers expanded the use of gene therapy in non-clinical studies to infectious diseases through the delivery of vectorized antibodies to well-defined antigens. Here, we further extend the utility of gene therapy beyond the "accepted" indications to include organophosphate poisoning. There are no approved preventives for the multi-organ damage resulting from acute or chronic exposure to organophosphates. We show that a single intramuscular injection of adeno-associated virus vector produces peak expression (~0.5 mg/ml) of active human butyrylcholinesterase (hBChE) in mice serum within 3–4 weeks post-treatment. This expression is sustained for up to 140 days post-injection with no silencing. Sustained expression of hBChE provided dose-dependent protection against VX in male and female mice despite detectable antibodies to hBChE in some mice, thereby demonstrating that expression of hBChE in vivo in mouse muscle is an effective prophylactic against organophosphate poisoning. [ABSTRACT FROM AUTHOR]

The engineered AAV-PHP.B family of adeno-associated virus efficiently delivers genes throughout the mouse central nervous system. To guide their application across disease models, and to inspire the development of translational gene therapy vectors for targeting neurological diseases in humans, we sought to elucidate the host factors responsible for the CNS tropism of the AAV-PHP.B vectors. Leveraging CNS tropism differences across 13 mouse strains, we systematically determined a set of genetic variants that segregate with the permissivity phenotype, and rapidly identified LY6A as an essential receptor for the AAV-PHP.B vectors. Interfering with LY6A by CRISPR/Cas9-mediated Ly6a disruption or with blocking antibodies reduced transduction of mouse brain endothelial cells by AAV-PHP.eB, while ectopic expression of Ly6a increased AAV-PHP.eB transduction of HEK293T and CHO cells by 30-fold or more. Importantly, we demonstrate that this newly discovered mode of AAV binding and transduction can occur independently of other known AAV receptors. These findings illuminate the previously reported species- and strain-specific tropism characteristics of the AAV-PHP.B vectors and inform ongoing efforts to develop next-generation AAV vehicles for human CNS gene therapy. [ABSTRACT FROM AUTHOR]

CRISPR-Cas is a powerful genome editing technology and has a great potential for in vivo gene therapy. Successful translational application of CRISPR-Cas to biomedicine still faces many safety concerns, including off-target side effect, cell fitness problem after CRISPR-Cas treatment, and on-target genome editing side effect in undesired tissues. To solve these issues, it is needed to design sgRNA with high cell-specific efficacy and specificity. Existing single-guide RNA (sgRNA) design tools mainly depend on a sgRNA sequence and the local information of the targeted genome, thus are not sufficient to account for the difference in the cellular response of the same gene in different cell types. To incorporate cell-specific information into the sgRNA design, we develop novel interpretable machine learning models, which integrate features learned from advanced transformer-based deep neural network with cell-specific gene property derived from biological network and gene expression profile, for the prediction of CRISPR-Cas9 and CRISPR-Cas12a efficacy and specificity. In benchmark studies, our models significantly outperform state-of-the-art algorithms. Furthermore, we find that the network-based gene property is critical for the prediction of cell-specific post-treatment cellular response. Our results suggest that the design of efficient and safe CRISPR-Cas needs to consider cell-specific information of genes. Our findings may bolster developing more accurate predictive models of CRISPR-Cas across a broad spectrum of biological conditions as well as provide new insight into developing efficient and safe CRISPR-based gene therapy. CRISPR-Cas is a powerful genome editing technology and has a great potential for in vivo gene therapy. To translate CRISPR-Cas into an efficient and safe therapeutic, it is critical to select target genes and design target-specific single guide RNAs such that they could maximize on-target in vivo efficiency as well as minimize the side effect induced by either off-target or on-target genome editing in undesired tissues. Due to experimental and clinical limitations, the CRISPR-Cas target efficiency and specificity in an intended condition (e.g. human) often need to be inferred from results in different conditions (e.g. animal model). This translational process imposes a big challenge in experimental design and potential risk in clinical development. To improve the cell-specific predictability of machine learning models and reveal important biological feature that determines the transferability of CRISPR-Cas9 across different cells, we develop an accurate and interpretable machine learning model that integrates features extracted from attention-based deep learning and knowledge-based cell-specific gene property. Our models significantly improve the performance of off-target specificity and cell-specific on-target efficiency prediction. We discover that network-based gene property is a key determinant of model predictability. Our finding may provide new insight into developing efficient and safe CRISPR-based gene therapy. [ABSTRACT FROM AUTHOR]

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD. [ABSTRACT FROM AUTHOR]

Emerging RNA-based approaches to disease detection and gene therapy require RNA sequences that fold into specific base-pairing patterns, but computational algorithms generally remain inadequate for these secondary structure design tasks. The Eterna project has crowdsourced RNA design to human video game players in the form of puzzles that reach extraordinary difficulty. Here, we demonstrate that Eterna participants’ moves and strategies can be leveraged to improve automated computational RNA design. We present an eternamoves-large repository consisting of 1.8 million of player moves on 12 of the most-played Eterna puzzles as well as an eternamoves-select repository of 30,477 moves from the top 72 players on a select set of more advanced puzzles. On eternamoves-select, we present a multilayer convolutional neural network (CNN) EternaBrain that achieves test accuracies of 51% and 34% in base prediction and location prediction, respectively, suggesting that top players’ moves are partially stereotyped. Pipelining this CNN’s move predictions with single-action-playout (SAP) of six strategies compiled by human players solves 61 out of 100 independent puzzles in the Eterna100 benchmark. EternaBrain-SAP outperforms previously published RNA design algorithms and achieves similar or better performance than a newer generation of deep learning methods, while being largely orthogonal to these other methods. Our study provides useful lessons for future efforts to achieve human-competitive performance with automated RNA design algorithms. [ABSTRACT FROM AUTHOR]

The dynamics of the cellular proportion of mutant mtDNA molecules is crucial for mitochondrial diseases. Cellular populations of mitochondria are under homeostatic control, but the details of the control mechanisms involved remain elusive. Here, we use stochastic modelling to derive general results for the impact of cellular control on mtDNA populations, the cost to the cell of different mtDNA states, and the optimisation of therapeutic control of mtDNA populations. This formalism yields a wealth of biological results, including that an increasing mtDNA variance can increase the energetic cost of maintaining a tissue, that intermediate levels of heteroplasmy can be more detrimental than homoplasmy even for a dysfunctional mutant, that heteroplasmy distribution (not mean alone) is crucial for the success of gene therapies, and that long-term rather than short intense gene therapies are more likely to beneficially impact mtDNA populations. [ABSTRACT FROM AUTHOR]

Both basic and translational research are continuously evolving, but the principles that underpin research integrity remain constant. These include rational, hypothesis-driven, and adequately planned and controlled science, which is carried out openly, honestly, and ethically. An important component of this should be minimising experimental irreproducibility. Biological systems, in particular, are inherently variable due to the nature of cells and tissues, as well as the complex molecules within them. As a result, it is important to understand and identify sources of variability and to strive to minimise their influence. In many instances, the application of metrology (the science of measurement) can play an important role in ensuring good quality research, even within biological systems that aren’t always amenable to many of the metrological concepts applied in other fields. Here, we introduce the basic concepts of metrology in relation to biological systems and promote the application of these principles to help avoid potentially costly mistakes in both basic and translational research. We also call on funders to encourage the uptake of metrological principles, as well as provide funding and support for later engagement with regulatory bodies. [ABSTRACT FROM AUTHOR]

The article describes de novo gene birth as the process that involves evolution of new genes from DNA sequences that were ancestrally non-genic. It notes that not all orphan genes arise de novo, and instead may emerge through fairly well-characterized mechanisms like gene duplication including retroposition or horizontal gene transfer followed by sequence. It adds that phylogenetic trees are limited by the set of closely related genomes that are available.

CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons. [ABSTRACT FROM AUTHOR]

Objectives: Duchenne muscular dystrophy (DMD) is a rare neuromuscular disorder that causes progressive weakness and early death. Gene therapy is an area of new therapeutic development. This qualitative study explored factors influencing parents’ and adult patients’ preferences about gene therapy. Methods: We report qualitative data from 17 parents of children with DMD and 6 adult patients. Participants responded to a hypothetical gene therapy vignette with features including non-curative stabilizing benefits to muscle, cardiac and pulmonary function; a treatment-related risk of death; and one-time dosing with time-limited benefit of 8–10 years. We used NVivo 11 to code responses and conduct thematic analyses. Results: All participants placed high value on benefits to skeletal muscle, cardiac, and pulmonary functioning, with the relative importance of cardiac and pulmonary function increasing with disease progression. More than half tolerated a hypothetical 1% risk of death when balanced against Duchenne progression and limited treatment options. Risk tolerance increased at later stages. Participants perceived a ‘right time’ to initiate gene therapy. Most preferred to wait until a highly-valued function was about to be lost. Conclusion: Participants demonstrated a complex weighing of potential benefits against harms and the inevitable decline of untreated Duchenne. Disease progression increased risk tolerance as participants perceived fewer treatment options and placed greater value on maintaining remaining function. In the context of a one-time treatment like gene therapy, our finding that preferences about timing of initiation are influenced by disease state suggest the importance of assessing ‘lifetime’ preferences across the full spectrum of disease progression. [ABSTRACT FROM AUTHOR]

The announcement of He Jiankui’s germline editing of human embryos has been followed by a torrent of almost universal criticism of the claim on scientific and ethical grounds. That criticism is warranted. There is little room for anything other than vociferous condemnation of He's announcement. Presenting the results of groundbreaking work by press conference and YouTube is not science. The issue now is not whether the work supporting the claims reported from China was done in an ethical manner. It was not. What is required to move forward is a justification for doing germline editing in humans. Many think there is none, and prohibitions abound. If such work is justifiable, a serious, rigorous framework must be imposed that insures that such research is done following the highest ethical standards that both protect human subjects and insure public trust and support. [ABSTRACT FROM AUTHOR]

Background: A major obstacle to using recombinant adenoviral vectors in gene therapy is the natural ability of human adenovirus to activate the classical and alternate complement pathways. These innate immune responses contribute to hepatic adenoviral uptake following systemic delivery and enhance the humoral immune responses associated with adenoviral infection. Methods: A recombinant Ad5 vector was genetically modified to display a peptide sequence (“rH17d’”), a known inhibitor of the classical complement pathway. The replication-defective vectors Ad5.HVR2-rH17d’ and Ad5.HVR5-rH17d’ were constructed by engineering the rH17d’ peptide into the hypervariable region (HVR)-2 or HVR5 of their major capsid protein hexon. Control Ad5 vectors were created by incorporation of a 6-histidine (His6)-insert in either HVR2 or HVR5 (Ad5.HVR2-His6 and Ad5.HVR5-His6, respectively). All vectors encoded CMV promoter-controlled firefly luciferase (Luc). The four vectors were evaluated in TIB76 mouse liver cells and immunocompetent mice to compare infectivity and liver sequestration, respectively. Results: In vitro studies demonstrated that preincubation of all the Ad5 vectors with fresh serum significantly increased their gene transfer relative to preincubation with PBS except Ad5.HVR5-rH17d’, whose infectivity of liver cells showed no serum-mediated enhancement. In line with that, mice injected with Ad5.HVR2-rH17d’ or Ad5.HVR5-rH17d’ showed significantly lower luciferase expression levels in the liver as compared to the respective control vectors, whereas efficiency of tumor transduction by rH17d’ and His6 vectors following their intratumoral injection was similar. Conclusions: Displaying a complement-inhibiting peptide on the Ad5 capsid surface by genetic modification of the hexon protein could be a suitable strategy for reducing Ad5 liver tropism (Ad5 sequestration by liver), which may be applicable to other gene therapy vectors with natural liver tropism. [ABSTRACT FROM AUTHOR]

Background: Advanced therapies are increasingly demanded by patients with the intent of treating some incurable conditions. Because family medicine professionals play an important role as health educators, their residency programs should incorporate new knowledge related to advanced therapies. To successfully implement these programs, how family medicine residents perceive these therapies should be investigated. The main components of perception, i.e. conceptual, procedural and attitudinal, refer to knowledge, skills and feelings, respectively. Methods and findings: We designed a specific questionnaire to assess the components of perceptions of advanced therapies in 300 medical residents enrolled in the Spanish National Family Medicine Residency Program. Each component consisted of 4 or 5 topics and each topic contained 6 items. Respondents scored highest in the procedural component (average 4.12±1.00), followed by the attitudinal (3.94±1.07) and conceptual component (3.04±1.43). Differences among the three components were statistically significant (p<0.00017). Family medicine residents perceived that procedures to implement advanced therapies are well established, especially their application. However, they felt their cognitive background was insufficient to respond efficiently to the expectations generated by these new therapeutic tools, especially in the regulatory framework. High awareness of the risks and limitations of these treatments was reflected by residents’ preference for clinically tested therapies. Although they appropriately situated treatment with these therapies within hospital care, they associated the biofabrication of novel products with research centers, although these therapeutic tools can be produced in different facilities. Conclusions: These results are potentially useful for designing future training programs and health policies for family medicine residents, and suggest the need to implement specific training programs in advanced therapies at the conceptual, procedural and attitudinal level. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) vectors represent promising candidates for gene therapy; however, pre-existing neutralizing antibodies (NAb) may reduce AAV vector delivery efficiency. In this study, the presence of AAV NAb was investigated in cats, which serve as a larger and outbred animal model for the prediction of gene therapy outcomes in humans but also in cats.Serum/plasma samples from 230 client-owned Swiss cats and 20 specified pathogen-free cats were investigated for NAb to AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9 using in vitro transduction inhibition and a beta-galactosidase assay. NAb to all tested AAV serotypes were found. Of the client-owned cats, 53% had NAb to one or more of the AAV serotypes. NAb (≥1:10) were found at frequencies of 5% (AAV6) to 28% (AAV7). The highest titers were found against AAV7 (≥1:160). The NAb prevalence to AAV2, AAV7 and AAV9 differed geographically. Regarding titers ≥1:10 against single AAV serotypes, age, breed and sex of the cats were not associated with the NAb prevalence. Cats with titers ≥1:20 against AAV2 and titers ≥1:40 against AAV7 were significantly younger than cats with low/no titers, and purebred cats were significantly more likely than non-purebred cats to have NAb to AAV2 (≥1:40). Additionally, regarding NAb to all AAV combined, female cats were significantly more likely than male cats to have NAb titers ≥1:40. Preliminary data using AAV-DJ indicated that less pre-existing NAb to the hybrid AAV-DJ can be expected compared to the wild-type AAV serotypes. AAV NAb will need to be taken into account for future in vivo gene therapy studies in cats. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is the most common type of malignant gliomas, characterized by genetic instability, intratumoral histopathological variability and unpredictable clinical behavior. Disappointing results in the treatment of gliomas with surgery, radiation and chemotherapy have fueled a search for new therapeutic targets and treatment modalities. Here we report new approach towards RNA interference therapy of glioblastoma multiforme based on the magnetic nanoparticles delivery of the double-stranded RNA (dsRNA) with homological sequences to mRNA of tenascin-C (TN-C), named ATN-RNA. The obtained nanocomposite consisted of polyethyleneimine (PEI) coated magnetic nanoparticles conjugated to the dsRNA show high efficiency in ATN-RNA delivery, resulting not only in significant TN-C expression level suppressesion, but also impairing the tumor cells migration. Moreover, synthesized nanomaterials show high contrast properties in magnetic resonance imaging (MRI) and low cytotoxicity combining with lack of induction of interferon response. We believe that the present work is a successful combination of effective, functional, non-immunostimulatory dsRNA delivery system based on magnetic nanoparticles with high potential for further application in GBM therapy. [ABSTRACT FROM AUTHOR]

The unique capacity of mesenchymal stem cells (MSCs) to migrate to the sites of damage, following intravenous transplantation, along with their proliferation and differentiation abilities make them promising candidates for MSC-based gene therapy. This therapeutic approach requires high efficacy delivery of stable transgenes to ensure their adequate expression in MSCs. One of the methods to deliver transgenes is via the viral transduction of MSCs. However, due to low transduction efficiency of MSCs, various polications are used to promote the association of viral particles with membranes of target cells. Among these polications polybrene is the most widely used one. Unfortunately, viral infection in presence of polybrene was shown to negatively affect proliferation rate of stem cells. The molecular mechanism underlying this effect is not yet uncovered. Therefore, the present study aimed to elucidate the mechanism of this phenomenon as well as to develop an effective approach to overcome the negative impact of polybrene on the properties of human endometrium-derived mesenchymal stem cells (hMESCs) during lentiviral infection. We found that the negative effect on proliferation observed during the viral infection in presence of polybrene is mediated by the polycation itself. Furthermore, we revealed that the treatment with polybrene alone led to the p38 MAPK-dependent premature senescence of hMESCs. These findings allowed us to develop an effective strategy to attenuate the negative polybrene impact on the hMESCs properties during lentiviral infection by inhibiting the activity of p38 MAPK. Importantly, the proposed approach did not attenuate the transduction efficiency of hMESCs, yet prevented polybrene-induced senescence and thereby restored the proliferation of the infected cells. These results provide the plausible means to reduce side effects of polybrene during the viral infection of primary cells, particularly MSCs. [ABSTRACT FROM AUTHOR]

The ability to produce large quantities of recombinant Adeno-Associated Virus (rAAV) vectors is an important factor for the development of gene therapy-based medicine. The baculovirus/insect cell expression system is one of the major systems for large scale rAAV production. So far, most technological developments concerned the optimization of the AAV rep and cap genes in order to be expressed correctly in a heterologous system. However, the effect of the baculovirus infection on the production of rAAV has not been examined in detail. In this study we show that the baculoviral cathepsin (v-CATH) protease is active on several (but not all) rAAV serotypes, leading to a partial degradation of VP1/VP2 proteins. Subsequently, we identified the principal v-CATH cleavage site in the rAAV8 capsid proteins and demonstrated that the cleavage is highly specific. The proteolytic degradation of VP1/VP2 AAV capsid proteins reduces the infectivity of rAAV vectors but can be prevented by the use of a baculovirus vector with a deletion of the chiA/v-cath locus or by addition of the E64 protease inhibitor during production. Moreover, the codon optimization of AAV cap performed for several serotypes and originally aimed at the removal of potential alternative initiation codons, resulted in incorporation of additional forms of truncated VP1 into the rAAV capsids. [ABSTRACT FROM AUTHOR]

Short and dysfunctional telomeres are sufficient to induce a persistent DNA damage response at chromosome ends, which leads to the induction of senescence and/or apoptosis and to various age-related conditions, including a group of diseases known as “telomere syndromes”, which are provoked by extremely short telomeres owing to germline mutations in telomere genes. This opens the possibility of using telomerase activation as a potential therapeutic strategy to rescue short telomeres both in telomere syndromes and in age-related diseases, in this manner maintaining tissue homeostasis and ameliorating these diseases. In the past, we generated adeno-associated viral vectors carrying the telomerase gene (AAV9-Tert) and shown their therapeutic efficacy in mouse models of cardiac infarct, aplastic anemia, and pulmonary fibrosis. Although we did not observe increased cancer incidence as a consequence of Tert overexpression in any of those models, here we set to test the safety of AAV9-mediated Tert overexpression in the context of a cancer prone mouse model, owing to expression of oncogenic K-ras. As control, we also treated mice with AAV9 vectors carrying a catalytically inactive form of Tert, known to inhibit endogenous telomerase activity. We found that overexpression of Tert does not accelerate the onset or progression of lung carcinomas, even when in the setting of a p53-null background. These findings indicate that telomerase activation by using AAV9-mediated Tert gene therapy has no detectable cancer-prone effects in the context of oncogene-induced mouse tumors. [ABSTRACT FROM AUTHOR]