Manufacturing of AAV vectors: translational challenges from development to industrialisation

AAV mediated gene therapy offers the potential for curative therapies for many fatal and debilitating diseases, however the translation of these concepts into licenced products is stymied by manufacturing challenges leading to very high costs and delayed commercialisation. This is an issue which vector developers need to address, specifically around productivities and vector purities including levels of empty non-functional capsids. To date vector developers have focused on the optimising of vector targeting and gene delivery, however there is an increasing need to recognise the issue of manufacturability of vectors, and to be able develop screening and assessment of novel vectors to ensure that in future innovations and development can be produced to the required quality and in the desired quantities and at an acceptable cost.

CRISPR/Cas9 has introduced new gene therapy possibilities for muscular dystrophies

Advancements in using CRISPR/Cas9 have introduced a host of new therapy possibilities for muscular dystrophies (MDs). There is a definite feeling of hope in the industry, but other barriers lay ahead, and they will define the future of MD gene editing. The ambiguity surrounding AAV transduction of satellite cells in vivo must be explained so that, if required, effort may be focused on optimizing vector targeting. Although the satellite cell correction needs are evident, it must be determined experimentally if high muscle turnover has a deleterious effect on CRISPR approaches. Another issue with muscular HDR is its low editing efficiency. Even outside the MD, exogenous, effective DNA integration would open up a slew of new possibilities.Either conventional HDR must be upgraded, or alternative techniques must be developed. The fact that both myotubes and latent satellite cells are post-mitotic means the latter are the most effective. Homology-independent targeted integration (HITI), homology-mediated end joining (HMEJ) and prime editing are three novel potentials. Duplication removal is another technique to restore full-length proteins. Duplications are the second most frequent DMD mutation, and a single sgRNA technique was used to restore dystrophin. To date, CRISPR/Cas9-mediated duplication removal has only been evaluated in DMD patient cells and must be tested in vivo. Because of their demonstrated track record in in vivo research and clinical trials, AAVs are expected to be employed in early generations of MD CRISPR therapy. Currently, AAVs may be the biggest choice, but future drugs will almost probably require a different delivery approach. It may take the shape of nanoparticles, which may carry a large range of transiently expressed payloads, while being very variable. If satellite cells can not be repaired, their capacity to escape immune reactions is crucial. To decrease the effects of muscle turnover, re-administration of nanoparticles may be utilized to treat MD throughout one's life. However, effective nanoparticle dosing for CRISPR in vivo editing has yet to be established in the muscle. Because this was not an AAV problem, the focus should be on new compositions of nanoparticles rather than improving the CRISPR/Cas9 system. The lack of published data suggests that nanoparticles' systemic muscle transport remains a considerable challenge. Due to muscle volume in the human body and the need to target muscles within the thoracic cavity, local intramuscular injections are not practical. Future research will focus primarily on developing an effective, muscle-specific nanoparticle that can be administered through circulation. The challenges ahead are tremendous, but with the appropriate focus and resources, answers will emerge, bringing therapeutic genome editing closer to the clinic than ever. While this research focused on DMD, the mentioned principles and methodology may and will undoubtedly be extended to several other MDs.

GENRE AND STYLISTIC PECULIARITIES OF SCIENTIFIC AND TECHNICAL TEXTS OF “SMART ENERGY SYSTEMS” PROFESSIOLECT IN MODERN ENGLISH LANGUAGE

Постановка задачи. Задача данной статьи - описать жанрово-стилистические особенности научно-технических текстов профессиолекта «Интеллектуальные энергетические системы» в современном английском языке. Результаты. Жанровое наполнение профессиолекта «Интеллектуальные энергетические системы» характеризуется широким синкретизмом и по своим системным признакам во многом напоминает полевую структуру, описание которой целесообразно через категориальную пару «центр vs. периферии». В связи с этим выделены 1) ядерные и 2) периферийные жанры научно-технических текстов профессиолекта «Интеллектуальные энергетические системы» с процентным соотношением 67,6% : 32,4 %. В качестве ядерных жанров выступают: 1) научно-техническая статья, 2) аннотация, 3) презентация и 4) доклад-отчёт. Периферийные жанры представлены: 1) стандартами на технологии и приборы; 2) инструктирующими текстами; 3) пресс-релизом. Классификационными критериями жанров научно-технических тестов, моделирующими их общую жанровую рамку, выступают: а) характер адресности; б) структурно-композиционная организация текста; в) репертуар языковых средств и их сочетание. Цельность жанровых образований в функциональном репертуаре «Интеллектуальные энергетические системы» (ИЭС) обусловлена тематическим единством. Рассмотренные научно-технические тексты представляют собой симбиоз языковых средств, принадлежащих разным семиотическим системам. Продуктивными синтаксическими моделями, соответствующими природе текстового материала профессиолекта ИЭС, выступают развернутые сложноподчиненные предложения с разветвленными придаточными структурами, сложносочиненные предложения с линейным характером связи, пассивные и безличные конструкции. Выводы. Научно-технические тексты профессиолекта «Интеллектуальные энергетические системы», представленные ядерными и периферийными жанрами, находятся в тесной содержательной взаимосвязи с исследованиями в сфере интеллектуальной энергетики. Коммуникация внутри исследуемого профессиолекта характеризуется прагматической направленностью, узковекторной адресностью и общностью институциональной принадлежности коммуникантов. Текстовые материалы отличаются нормативностью отбора и комбинации языковых средств и высокой стереотипностью синтаксических структур. Наряду с признаками, объединяющими все вышеперечисленные жанры, каждый из них выполняет собственные, присущие лишь ему стилистические задачи, демонстрируя разноплановый характер выразительных средств и содержательное наполнение. Statement of the problem. The purpose of this article is to describe the genre and stylistic peculiarities of the scientific and technical texts of the "Smart Energy Systems" professiolect in modern English. Results. The genre content of the "Smart Energy Systems" professiolect is characterized by broad syncretism and in terms of its systemic features resembles a field structure, which study is advisable to conduct using the categorical pair “center vs. periphery". In this regard nuclear and peripheral genres of scientific and technical texts of the "Smart Energy Systems" professiolect were distinguished with a percentage ratio of 67.6% : 32.4%. The nuclear genres are: 1) scientific and technical article, 2) abstract, 3) presentation and 4) report. Peripheral genres are represented by: 1) standards for technologies and equipment; 2) instructional texts: manuals for specialists and consumers; 3) press release. The classification criteria of scientific and technical tests, modeling their general genre framework, are: a) the nature of the target audience; b) structural and compositional organization of the text; c) the repertoire of linguistic tools and their combination. The integrity of genre formations in the "Smart Energy Systems" functional repertoire is characterized by its thematic unity. The considered scientific and technical texts represent a symbiosis of linguistic resources belonging to different semiotic systems. Productive syntactic models that correspond to the nature of the textual material of the "Smart Energy Systems" professiolect are: detailed complex sentences with subordinate structures, compound sentences with a linear connection, passive and impersonal constructions. Conclusion. Scientific and technical texts of the professiolect "Intelligent Energy Systems", represented by nuclear and peripheral genres, are closely related to research in the field of smart power industry. Communication within the studied professiolect is characterized by a pragmatic orientation, narrow-vector targeting and common professional interests of the communicants. The material is distinguished by the normative selection and combination of linguistic tools and high stereotype of syntactic structures. Along with features that unite all genres, each of them performs its own stylistic tasks, demonstrating the diverse nature of expressive tools and content.

In vivo lentiviral vector gene therapy to cure hereditary tyrosinemia type 1 and prevent development of precancerous and cancerous lesions

Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) delays but in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate for the first time a complete cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase (FAH) transgene in the porcine model of HT1. This therapy was well tolerated and provided stable long-term expression of FAH in pigs with HT1. Genomic integration displayed a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach.

Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum)

Cichorium intybus var. foliosum (witloof) is an economically important crop with a high nutritional value thanks to many specialized metabolites, such as polyphenols and terpenoids. However, witloof plants are rich in sesquiterpene lactones (SL) which are important for plant defense but also impart a bitter taste, thus limiting industrial applications. Inactivating specific genes in the SL biosynthesis pathway could lead to changes in the SL metabolite content and result in altered bitterness. In this study, a CRISPR/Cas9 genome editing workflow was implemented for witloof, starting with polyethylene glycol (PEG) mediated protoplast transfection for CRISPR/Cas9 vector delivery, followed by whole plant regeneration and mutation analysis. Protoplast transfection efficiencies ranged from 20 to 26 %. A CRISPR/Cas9 vector targeting the first exon of the phytoene desaturase (CiPDS) gene was transfected into witloof protoplasts and resulted in the knockout of CiPDS, giving rise to an albino phenotype in 23% of the regenerated plants. Further implementing our protocol, the SL biosynthesis pathway genes germacrene A synthase (GAS), germacrene A oxidase (GAO), and costunolide synthase (COS) were targeted in independent experiments. Highly multiplex (HiPlex) amplicon sequencing of the genomic target loci revealed plant mutation frequencies of 27.3, 42.7, and 98.3% in regenerated plants transfected with a CRISPR/Cas9 vector targeting CiGAS, CiGAO, and CiCOS, respectively. We observed different mutation spectra across the loci, ranging from consistently the same +1 nucleotide insertion in CiCOS across independent mutated lines, to a complex set of 20 mutation types in CiGAO across independent mutated lines. These results demonstrate a straightforward workflow for genome editing based on transfection and regeneration of witloof protoplasts and subsequent HiPlex amplicon sequencing. Our CRISPR/Cas9 workflow can enable gene functional research and faster incorporation of novel traits in elite witloof lines in the future, thus facilitating the development of novel industrial applications for witloof.

EGFR-Binding Peptides: From Computational Design towards Tumor-Targeting of Adeno-Associated Virus Capsids

The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the EGFR dimerization interface. Two new peptides and a reference (EDA peptide) were chemically synthesized, and their binding ability characterized. Presentation of these peptides in each of the 60 capsid proteins of recombinant adeno-associated viruses (rAAV) via a genetic based loop insertion enabled targeting of EGFR overexpressing tumor cell lines. Furthermore, tissue distribution and tumor xenograft specificity were analyzed with systemic injection in chicken egg chorioallantoic membrane (CAM) assays. Complex correlations between the targeting of the synthetic peptides and the viral vectors to cells and in ovo were observed. Overall, these data demonstrate the potential of computational design in combination with rational capsid modification for viral vector targeting opening new avenues for viral vector delivery and specifically suicide gene therapy.

Proteomics Analysis Identified Hub Genes Associated With RNAi-Induced Silencing of G1 Cyclins in MDA-MB-231 Cells

Background: The G1 cyclins are the most potent candidates in the pathogenesis of breast cancer. This study was designed to analyze the synergistic effect of G1 cyclins silencing on the proliferation of breast cancer cells and to identify G1 cyclins-molecular targets by proteomics approach.Methods: The MDA-MB-231 cells were transfected by a dual shRNA vector targeting G1 cyclins through a bidirectional survivin promoter. Silencing efficacy and cell proliferation were evaluated by real-time PCR, Western blot, and MTS assays, respectively. The protein expression profile was evaluated by 2D gel electrophoresis and mass spectroscopy. Further, bioinformatics tools were applied to identify the molecular targets by G1 cyclins and their possible biological consequences.Results: In response to G1 cyclins silencing, the proliferation of cells exposed to the dual shRNA vector decreased significantly at 72 h post-transfection. The reduction of G1 cyclins proteins was following their mRNA expression level as well. Protein signature of cells was altered in response to the silencing of G1 cyclins, 13 up-regulated, and seven down-regulated. Network analysis of G1 cyclins-regulated proteins identified ACTB, HSP90AA1, ALB, and HSPA5 as the hub genes according to the degree method.The regulated proteins by G1 cyclins participate in cancer-related pathways such as PI3K-Akt signaling pathway and pathways in cancer (HSP90AA1; HSP90AB1; HSP9B1), HIF-1 signaling pathway (LDHA; ALDOA; PGK1), apoptosis and proteoglycans in cancer (ACTB).Conclusions: We identified the G1 cyclins-regulated proteins and their mode of action in the pathogenesis of breast cancer. Our findings suggested that G1 cyclins participate in various biological functions. Meanwhile, it offers a new perception of the interactions among G1 cyclins-regulated proteins to identify targeted treatment. It follows-up research in the field of breast cancer.

Suppression of Gremlin by RNA interference attenuates glucose-induced fibrogenesis in rat mesangial cells

Background: diabetic nephropathy (DN) is the most common cause of end-stage of renal disease. It is beneficial for us to find effective way to treat the disease. Gremlin is deemed as a key factor in the development of diabetic nephropathy at present. We hypothesized that the pathological changes might be prevented by eliminating Gremlin function in high glucose-induced renal fibrosis. Methods: lentiviral vector targeting Gremlin was applied to inhibit Gremlin expression at a high glucose concentration which simulated diabetic nephropathy in rat mesangial cells. Results: the shRNA vector, designated shGremlin, significantly inhibited Gremlin expression in rat mesangial cells cultured under high glucose conditions. Increase in BMP-7 as well as its downstream genes or proteins including phospholated Smad 1/5/8, type IV collagen and fibronectin was observed. Conclusions: our work provides a valuable method to prevent glomerular/renal fibrosis with RNA interference, and also enable development of new therapies that target Gremlin.

Preparation and Evaluation of Upconversion Nanoparticles Based miRNA Delivery Carrier in Colon Cancer Mice Model

Therapeutic efficacy of solid tumor is often severely hampered by poor penetration of therapeutics into diseased tissues and lack of tumor targeting. In this study, the functionalized upconversion nanoparticles (UCNP)-based delivery vector targeting cancer cells was developed. Firstly, NaYF4:Yb/Tm (UCNP) was prepared with the solvothermal method for the uniform nanoparticle size and brilliant lattice structure. The SiO2 coated UCNP was demonstrated a high upconversion emission and good monodispersity, which was coupled with polyetherimide (PEI) and miR-145 vector. Then, it was further functionalized via hyaluronic acid (HA) (UCNP/PEI/HA Nanocomplex, UCNPs) coating for the targeted delivery and improved biocompatibility. The UCNPs/miR-145 displays an excellent biocompatibility, a high level of cellular uptake and miR-145 expression, which results in a significant cell cycle arrest in G1, and induces CCND1, CDK6 and CCNE2 proteins downregulation. In vivo, the HA-coated UCNPs were enriched at the tumor site by targeting and retention effects, which resulted in a significant inhibition of tumor growth. Histological experiments demonstrated that UCNPs did not show significant toxicity in mice colon cancer model. Taken together, a UCNPs-based delivery platform was successfully constructed and used for miRNA target delivery, which provided a new method and idea for bioengineering and nanotechnology-based tumor therapy.