Vectorology - GEG Tech top picks

Collagen VI-related dystrophies (COL6-RD) are caused by mutations in the COL6A1, COL6A2 and COL6A3 genes that encode the alpha chains of collagen VI, a key component of the extracellular matrix. The prevalent pathogenic mutation in COL6A1 is a single nucleotide substitution, where a glycine is replaced by an arginine in the N-terminus of the triple helix domain which affects the folding of the resulting protein, hindering the association of tetramers to create the necessary collagen VI microfibrils. Moreover, there is currently no effective treatment available for COL6-RD. However, a recent paper demonstrates the use of CRISPR-Cas9 gene editing to mitigate the pathogenic effects of a dominant negative mutation in the COL6A1 gene. Nevertheless, an obvious hurdle for this type of therapy is the delivery of CRISPR-Cas9 reagents to fibroblasts in vivo; successful editing of somatic tissues such as muscle remains a significant challenge in the broader field. The research team hopes that recent advances in several areas of CRISPR delivery vehicles, including muscle adeno-associated trophic virus vectors and tissue-specific nanoparticles, may be the answer.

Optogenetics may enable mutation-independent, circuit-specific restoration of neuronal function in neurological diseases. Retinitis pigmentosa is a neurodegenerative eye disease where loss of photoreceptors can lead to complete blindness. In a blind patient, the researchers combined intraocular injection of an adeno-associated viral vector encoding ChrimsonR with light stimulation via engineered goggles. 

The Viral Vectors and Plasmid DNA Manufacturing Market (2nd Edition), 2018-2030 report offers a comprehensive study of the current scenario of manufacturing of viral and non-viral vectors that are primarily used for the development of gene therapies and T-cell therapies. The study features an in-depth analysis, highlighting the capabilities of a diverse set of players, covering both contract manufacturers and companies with in-house capabilities.

Here the authors review the factors related to risk assessment regarding viral vector usage in animals and the relevant regulatory documents associated with this research, and they highlight the most commonly used viral vectors in research today. This review is particularly focused on the background, use in research and associated health and environmental risks related to adenoviral, adeno-associated viral, lentiviral, and herpesviral vectors.

Researchers have successfully used a viral transport mechanism to deliver a gene to the brain of mice that prevented the development of Alzheimer’s disease (AD). The results could have a profound impact on the future of gene therapy in treating Alzheimer’s.

In neurology, It is critical to be able to transfer genes  in the primate brain to the study of neural connectivities and neural circuits. In this review, the authors summarize their current achievements as follows

1) They compared the features of gene transfer using five different AAV serotypes in combination with three different promoters,

2) They used target-specific double-infection techniques in combination with TET-ON and TET-OFF using lentiviral retrograde vectors for enhanced visualization of neural connections.

3) They used an AAV-mediated gene transfer method to study the transcriptional control for amplifying fluorescent signals using the TET/TRE system in the primate neocortex.

Hookipa is starting its first clinical trial for a vaccine against human cytomegalovirus. This is the leading candidate of its platform for novel viral vectors, which promises to yield better vaccines for infections and cancer.

In this paper, the authors succinctly review these fast-paced developments and technologies, highlighting their relative merits and potential bottlenecks, when used as part of in vivo and ex vivo gene-editing strategies.

Here, the authors look back at delivery strategies having been used in the in vivo delivery of ZFN, TALEN and CRISPR/Cas9 system, followed by methodologies currently undergoing testing in clinical trials, and potential delivery strategies provided by analyzing characteristics of nucleases and commonly used vectors.

The researcher has developed rabies virus-like particles (RV-VLPs) with immunogenic features expressed in mammalian cells for vaccine applications. In this chapter the methods to obtain and characterize a stable HEK293 cell line expressing RV-VLPs are detailed and the authors also describe  immunization protocols to study the immunogenicity of RV-VLPs.

Here, the scientists highlight prominent tumorigenic risks of iPSC products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration.They demonstrate that combined transgene-free (TGF) reprogramming using nonintegrating Sendai viral vectors and enzymatic dissociation allow teratoma-free transplantation of iPSC progeny in the most rigorous mouse model in testing the tumorigenicity of iPSC products.Further long-term safety assessment and improvement in TGF-iPSC specification into a mature b-cell phenotype would lead to customized safe islet replacement therapy for type 1 diabetes.

Conventional gammaretroviral vectors achieved successful results in clinical trials: treated patients had therapeutic gene expression in target cells and had improved symptoms of diseases. However, serious side-effects of leukemia occurred, caused by retroviral insertional mutagenesis. The authors discuss about these results and about the design of new tools for gene therapy.

Low-density lipoprotein (LDL) can carry thousands of fat molecules,
such as cholesterol, and high levels of LDL in the blood are clinically associated with an increased risk of cardiovascular disease or atherosclerosis. By installing a point mutation through gene editing in the gene encoding an enzyme called PCSK9 that is involved in the uptake of LDL cholesterol from the blood into cells, an international team of researchers led by the University of Zurich succeeded in permanently lowering high levels of LDL cholesterol in the blood of mice and macaques. To control the delivery of the basic editing tool into the animals' livers, the researchers adapted the RNA technology used in the COVID-19 vaccines. However, instead of encapsulating an RNA encoding the SARS-CoV2 spike protein in lipid nanoparticles, they encapsulated an RNA encoding the adenine base editor. This study opens the possibility of curing patients with inherited metabolic liver diseases.

Oxford BioMedica, has signed a fifteen year lease on a new facility at its site in Oxford, UK-based, which when operational will more than double the firm’s bioprocessing capacity.

The 7,800m2 facility – the fourth plant at the site – will contain four GMP clean room suites and two fill and finish suites for lentiviral vector production and will help feed demand for a market the firm expects to grow to $800 million by 2026.

Lentiviral vectors (LVs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies.  The authors of this study have previously reported successful, transient and stable production of LVs pseudotyped with RD114-TR for good transduction of T lymphocytes and CD34+ cells. In this work, to simplify the vector design, they decided to codon-optimize the entire RD114-TR ORF. In fact, the elimination of the interfering sequences would have avoided using the BGI, therefore reducing the size of the vector. Unexpectedly, they found that, despite the high level of transcription/translation and cytosol export, RD114-TRco is functionally dead. These data strengthen the conclusion, also supported by other studies, that codon optimization may not always lead to functional improvement of the gene of interest.

Here, the scientists assessed the effect of passive immunization against α-syn in a new mouse model of axonal transport and accumulation of α-syn. For these purpose, non-transgenic, α-syn knock-out and mThy1-α-syn tg (line 61) mice received unilateral intra-cerebral injections with a lentiviral (LV)-α-syn vector construct followed by systemic administration of the monoclonal antibody 1H7 (recognizes amino acids 91-99) or control IgG for 3 months. Unilateral LV-α-syn injection resulted in axonal propagation of α-syn in the contra-lateral site with subsequent behavioral deficits and axonal degeneration. Passive immunization with 1H7 antibody reduced the axonal accumulation of α-syn in the contra-lateral side and ameliorated the behavioral deficits. Together this study supports the notion that immunotherapy might improve the deficits in models of synucleinopathy by reducing the axonal propagation and accumulation of α-syn. This represents a potential new mode of action through which α-syn immunization might work.

A greater number and variety of workers will be exposed to commercial synthetic biology risks in the future, including risks to a variety of workers from the use of lentiviral vectors as gene transfer devices. There is a need to review and enhance current protection measures in the field of synthetic biology, whether in experimental laboratories where new advances are being researched, in health care settings where treatments using viral vectors as gene delivery systems are increasingly being used, or in the industrial bioeconomy.

In the mid-1990s, several years after a variety of viral vectors started being used for gene transfer into cells, tissues, and in some cases humans, it became clear that there were considerable limitations. For applications requiring a stable genetic modification that could lead to sustained gene expression in cells and their progeny, a delivery vehicle was needed that could transduce foreign cargo into dividing and nondividing cells, without causing immuno- or genotoxicity. A decade earlier, human immunodeficiency virus (HIV) had been identified as the cause of AIDS, and rapid studies of its biology led to the idea that this genus of retrovirus—lentiviruses—could be optimized for gene therapy.

This review focuses on CARs recognizing the B cell antigen CD19. Both retro- and lentiviral vectors are used encoding the different anti-CD19 CAR constructs comprising costimulatory molecules like CD28, CD137/4-1BB and OX40 either alone (2nd generation CARs) or in combination (3rd generation CARs). Current up-to-date published studies on anti-CD19 CAR therapy for acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and Non-Hodgkin lymphoma (NHL) with observed side effects are discussed and an outlook on 59 ongoing trials is given.

Here the authors tested the ability of the lentiviral vector to generate stable cell line without transduction step. Indeed, they observe that a co-transfection of a Human Embryonic Kidney (HEK) 293 packaging cell line with Gag–pol (GP) and a transfer vector, without the envelope vector, produces a stable cell line after 2 weeks of selection. They conclude that if the packaging cell is also the target cell, then gene integration leading to a stable cell line can be accomplished without viral particle infection.

Toca FC (extended-release 5-fluorocytosine) and Toca 511 (vocimagene amiretrorepvec) are an investigational therapeutic combination for glioma, consisting of two parts: a prodrug that is inactive on its own and a modified virus that infects the tumor and delivers an enzyme, which then activates the drug and allows it to kill the glioma cells. Cloughesyet al. tested this therapy in 45 human patients with recurrent or progressive high-grade glioma and discovered that the treatment was well tolerated and improved survival compared to an external control group. In addition, the authors identified a gene signature that correlated with response to the treatment, which may help identify the patients most likely to benefit from this approach.

In this review, the authors discuss existing and emerging strategies for directing genetic manipulations to targeted neurons in the adult primate central nervous system. They review the literature on viral vectors for gene delivery to neurons, focusing on adeno-associated viral vectors and lentiviral vectors, their tropism for different cell types, and prospects for new variants with improved efficacy and selectivity.