Human Gene Therapy Methods最新文献

Adeno-associated virus vectors are a powerful tool for gene transfer approaches. We have established a simple and fast plasmid-based production system for achieving high adeno-associated virus titers within 6 working days. The same procedure can be used for all serotypes and thus allows direct comparability of different serotypes. In this protocol we describe a step-by-step procedure that results in well-characterized vectors suitable for both in vitro approaches and preclinical studies.

A set of vectors for Cre recombinase-dependent expression of the hybrid suicidal FCU1 transgene was constructed, including a two-plasmid system wherein the FCU1 and Cre transgenes reside in separate vectors, and single-plasmid variants in which a single plasmid bears both transgenes. To improve the safety profile and specificity in cancer gene therapy applications, as well as to ensure stable propagation of plasmids in bacterial cells, the Cre/LoxP system components were optimized. A bicistronic vector with the Cre expression cassette placed between the LoxP sites unidirectionally with FCU1 cDNA resulted in higher therapeutic efficiency compared with the double-plasmid system in an enzyme-prodrug suicide cancer gene therapy scheme. Therefore, the feasibility of a single-plasmid approach in the development of cancer gene therapy with hierarchical enhancement of therapeutic transgene expression has been demonstrated.

The requirement for robust analytical methods to characterize adeno-associated virus (AAV) vectors is immediate, as the field advances more AAV gene therapies into the clinic and onto commercialization. AAV capsid proteins (VPs) are critical for viral infectivity and vector potency. Thus, complete characterization of the constituent viral capsid proteins of AAV vectors, including their sequences and post-translational modifications (PTMs), is highly recommended to ensure AAV product quality and consistency. Typically, SDS-PAGE analysis followed by in-gel enzymatic digestion and liquid chromatography/tandem mass spectrometry (LC/MS/MS) is used for the characterization of viral capsid proteins. However, due to the limited recovery of digested peptides from the gel, determination of N-terminal sequences of VPs has not been reported to date. In this study, a direct liquid chromatography/mass spectrometry (LC/MS) intact protein analysis was developed to characterize viral capsid proteins in a variety of AAV serotypes. Both N- and C-terminal sequences of six AAV serotypes have been identified based on accurate mass measurement. This method can be used to confirm the identity of AAV serotype and monitor potential capsid protein heterogeneity. Complete sequence confirmation of AAV2 VPs was achieved through LC/MS/MS analysis of peptides generated using multiple enzymatic digestions. LC/MS/MS analysis confirmed the sequences for both N- and C-termini of capsid VPs and revealed acetylation on the N-termini of VP1 and VP3, consistent with LC/MS intact protein analysis.

Adeno-associated virus (AAV) inverted terminal repeats (ITRs) are key elements of AAV. These guanine-cytosine-rich structures are involved in the replication and encapsidation of the AAV genome, along with its integration in and excision from the host genome. These sequences are the only AAV-derived DNA sequences conserved in recombinant AAV (rAAV), as they allow its replication, encapsidation, and long-term maintenance and expression in target cells. Due to the original vector design, plasmids containing the gene of interest flanked by ITRs and used for rAAV production often present incomplete, truncated, or imperfect ITR sequences. For example, pSUB201 and its derivatives harbor a truncated (14 nt missing on the external part of the ITR), flop-orientated ITR plus 46 bp of non-ITR viral DNA at each end of the rAAV genome. It has been shown that rAAV genomes can be replicated, even with incomplete, truncated, or imperfect ITR sequences, leading to the production of rAAV vectors in transfection experiments. Nonetheless, it was hypothesized that unmodified wild-type (WT) ITR sequences could lead to a higher yield of rAAV, with less non-rAAV encapsidated DNA originating from the production cells and/or baculovirus shuttle vector genomes. This work studied the impact of imperfect ITRs on the level of encapsidated rAAV genomes and baculovirus-derived DNA sequences using the baculovirus/Sf9 cells production system. Replacement of truncated ITRs with WT and additional wtAAV2 sequences has an impact on the two major features of rAAV production: (1) a rise from 10% to 40% of full capsids obtained, and (2) up to a 10-fold reduction in non-rAAV encapsidated DNA. Furthermore, this study considered the impact on these major parameters of additional ITR elements and ITRs coupled with various regulatory elements of different origins. Implementation of the use of complete ITRs in the frame of the baculovirus-based rAAV expression system is one step that will be required to optimize the quality of rAAV-based gene therapy drugs.

The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described. Numerous controls are included in order to monitor the technical procedure.

Gene therapy is a rapidly evolving field. So far, there have been >2,400 gene therapy products in clinical trials and four products on the market. A prerequisite for producing gene therapy products is ensuring their quality and safety. This requires appropriately controlled and standardized production and testing procedures that result in consistent safety and efficacy. Assuring the quality and safety of lentivirus-based gene therapy products in particular presents a great challenge because they are cell-based multigene products that include viral and therapeutic proteins as well as modified cells. In addition to the continuous refinement of a product, changes in production sites and manufacturing processes have become more and more common, posing challenges to developers regarding reproducibility and comparability of results. This paper discusses the concept of developing a first World Health Organization International Standard, suitable for the standardization of assays and enabling comparison of cross-trial and cross-manufacturing results for this important vector platform. The standard will be expected to optimize the development of gene therapy medicinal products, which is especially important, given the usually orphan nature of the diseases to be treated, naturally hampering reproducibility and comparability of results.

Multimodal lentiviral vectors (LVs) allow switching between constitutive and tetracycline-regulated gene co-expressions in genetically modified cells. Transduction of murine primary hematopoietic progenitor cells (HPCs) with multimodal LVs in the absence of doxycycline ensures the constitutive expression of gene of interest 1 (GOI1) only. In the presence of doxycycline, induced tetracycline-regulated expression of a second GOI (GOI2) allows evaluation of the collaboration between two genes. Drug removal retains constitutive expression, which allows the contribution of an individual gene into created networks to be studied. Doxycycline-dependent switching can be tracked via fluorescent markers coupled to constitutive and tetracycline-regulated GOIs. This article describes transduction of murine primary HPCs with different doses of multimodal LVs, distinct cytokine conditions, and their influence on the number and viability of cells co-expressing both collaborating GOIs upon doxycycline induction. A 2-week protocol is provided for multimodal LV production, titer determination, and evaluation of tetracycline responsive promoter background activity in a murine fibroblast cell line. The power of this model to assess the dose/time/order-controlled contribution of single and multiple genes into hematopoietic networks opens new routes in reprogramming, stem cell, and leukemia biology.

Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is nonpathogenic, and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy. The focus is on European regulations and how they are administered in the United Kingdom, although many of the principles will be similar for other regions, including the United States. The report justifies the rationale for using third-generation lentiviral vectors to achieve gene delivery for in vivo and ex vivo applications; briefly summarizes the extant regulatory guidance for gene therapies, categorized as advanced therapeutic medicinal products (ATMPs); provides guidance on specific regulatory issues regarding gene therapies; presents an overview of the key stakeholders to be approached when pursuing clinical trials authorization for an ATMP; and includes a brief catalogue of the documentation required to submit an application for regulatory approval of a new gene therapy.

The RNA interference pathway is an evolutionary conserved post-transcriptional gene regulation mechanism that is exclusively triggered by double-stranded RNA inducers. RNAi-based methods and technologies have facilitated the discovery of many basic science findings and spurred the development of novel RNA therapeutics. Transient induction of RNAi via transfection of synthetic small interfering RNAs can trigger the selective knockdown of a target mRNA. For durable silencing of gene expression, either artificial short hairpin RNA or microRNA encoding transgene constructs were developed. These miRNAs are based on the molecules that induce the natural RNAi pathway in mammals and humans: the endogenously expressed miRNAs. Significant efforts focused on the construction and delivery of miRNA cassettes in order to solve basic biology questions or to design new therapy strategies. Several viral vectors have been developed, which are particularly useful for the delivery of miRNA expression cassettes to specific target cells. Each vector system has its own unique set of distinct properties. Thus, depending on the specific application, a particular vector may be most suitable. This field was previously reviewed for different viral vector systems, and now the recent progress in the field of miRNA-based gene-silencing approaches using lentiviral vectors is reported. The focus is on the unique properties and respective limitations of the available vector systems for miRNA delivery.