Intestinal Transgene Delivery with Native E. coli Chassis Allows Persistent Physiological Changes

Live bacterial therapeutics (LBT) could reverse disease by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally-raised (CR) hosts have been unsuccessful, because engineered microbial organisms (i.e., chassis) cannot colonize the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli isolated from stool cultures of CR mice were modified to express functional bacterial (bile salt hydrolase) and eukaryotic (Interleukin-10) genes. Reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect host physiology and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to knock-in specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts, and enables LBT with curative intent.

Tagging and Capturing of Lentiviral Vectors Using Short RNAs

Lentiviral (LV) vectors have emerged as powerful tools for transgene delivery ex vivo but in vivo gene therapy applications involving LV vectors have faced a number of challenges, including the low efficiency of transgene delivery, a lack of tissue specificity, immunogenicity to both the product encoded by the transgene and the vector, and the inactivation of the vector by the human complement cascade. To mitigate these issues, several engineering approaches, involving the covalent modification of vector particles or the incorporation of specific protein domains into the vector’s envelope, have been tested. Short synthetic oligonucleotides, including aptamers bound to the surface of LV vectors, may provide a novel means with which to retarget LV vectors to specific cells and to shield these vectors from neutralization by sera. The purpose of this study was to develop strategies to tether nucleic acid sequences, including short RNA sequences, to LV vector particles in a specific and tight fashion. To bind short RNA sequences to LV vector particles, a bacteriophage lambda N protein-derived RNA binding domain (λN), fused to the measles virus hemagglutinin protein, was used. The λN protein bound RNA sequences bearing a boxB RNA hairpin. To test this approach, we used an RNA aptamer specific to the human epidermal growth factor receptor (EGFR), which was bound to LV vector particles via an RNA scaffold containing a boxB RNA motif. The results obtained confirmed that the EGFR-specific RNA aptamer bound to cells expressing EGFR and that the boxB containing the RNA scaffold was bound specifically to the λN RNA binding domain attached to the vector. These results show that LV vectors can be equipped with nucleic acid sequences to develop improved LV vectors for in vivo applications.

Efficient Targeted Transgene Delivery to Injured Lower Motor Neurons

Peripheral nerve injuries yield devastating consequences, and surgical repair outcomes remain suboptimal. Novel therapeutic strategies such as gene therapy could improve peripheral nerve regeneration. Though adeno-associated virus (AAV) vectors have delivered transgenes to intact peripheral neurons, transduction of transected neurons relevant to management of peripheral nerve injuries has not been reported. Herein, in vivo transduction efficiency of axotomized murine facial neurons using four AAV capsids packaging a fluorescent reporter transgene, tdTomato, is characterized. Proximal stumps of transected facial nerve branches in C57Bl/6J mice were immersed in AAV solutions. Four weeks later, facial motor nuclei were volume-imaged via whole-mount two-photon excitation microscopy, and machine learning-based image segmentation quantified the proportion of transgene expressing neurons. We observed remarkable retrograde transduction efficiency with AAV-PHP.S and AAV-F, with expression levels sufficient to detect intrinsic tdTomato fluorescence. This study confirms successful in vivo retrograde transgene delivery to transected peripheral neurons, an approach that carries potential as a research tool and future therapeutic strategy.

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

Vectors developed from adeno-associated virus (AAV) are powerful tools for in vivo transgene delivery in both humans and animal models, and several AAV-delivered gene therapies are currently approved for clinical use. However, AAV-mediated gene therapy still faces several challenges, including limited vector packaging capacity and the need for a safe, effective method for controlling transgene expression during and after delivery. Riboswitches, RNA elements which control gene expression in response to ligand binding, are attractive candidates for regulating expression of AAV-delivered transgene therapeutics because of their small genomic footprints and non-immunogenicity compared to protein-based expression control systems. In addition, the ligand-sensing aptamer domains of many riboswitches can be exchanged in a modular fashion to allow regulation by a variety of small molecules, proteins, and oligonucleotides. Riboswitches have been used to regulate AAV-delivered transgene therapeutics in animal models, and recently developed screening and selection methods allow rapid isolation of riboswitches with novel ligands and improved performance in mammalian cells. This review discusses the advantages of riboswitches in the context of AAV-delivered gene therapy, the subsets of riboswitch mechanisms which have been shown to function in human cells and animal models, recent progress in riboswitch isolation and optimization, and several examples of AAV-delivered therapeutic systems which might be improved by riboswitch regulation.

Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment

Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy likely due to the cells’ heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette.Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency.

Planet of the AAVs: The Spinal Cord Injury Episode

The spinal cord injury (SCI) is a medical and life-disrupting condition with devastating consequences for the physical, social, and professional welfare of patients, and there is no adequate treatment for it. At the same time, gene therapy has been studied as a promising approach for the treatment of neurological and neurodegenerative disorders by delivering remedial genes to the central nervous system (CNS), of which the spinal cord is a part. For gene therapy, multiple vectors have been introduced, including integrating lentiviral vectors and non-integrating adeno-associated virus (AAV) vectors. AAV vectors are a promising system for transgene delivery into the CNS due to their safety profile as well as long-term gene expression. Gene therapy mediated by AAV vectors shows potential for treating SCI by delivering certain genetic information to specific cell types. This review has focused on a potential treatment of SCI by gene therapy using AAV vectors.

Current Non-Viral Gene Therapy Strategies for the Treatment of Glioblastoma

Background: Glioblastoma constitutes the most frequent and aggressive primary malignant brain tumor in adults. Despite the advances in its treatment, its prognosis remains very poor. Gene therapy has been proposed as a complementary treatment, since it may overcome the problem of the blood-brain barrier for systemic therapies, allowing to target tumor cells and their tumor microenvironment locally, without affecting the normal brain parenchyma. In comparison with viral vectors, non-viral vectors became an attractive tool due to their reduced potential of biosafety risks, lower cost, higher availability and easy storage. Objective: In this article, we aimed to outline the current preclinical and clinical developments of non-viral delivery systems for therapeutic transgene delivery in malignant gliomas. Conclusion: Non-viral vectors are efficient tools for gene delivery since they exhibit reduced non-specific cytotoxicity and can go through several modifications in order to achieve high tumor tropism and the ability to cross the blood-brain barrier to access the tumor mass. However, further evaluations in preclinical models and clinical trials are required in order to translate it into the neuro-oncology clinic.

Transgene Delivery to Human Induced Pluripotent Stem Cells Using Nanoparticles

Human induced pluripotent stem cells (hiPSCs) and hiPSCs-derived cells have the potential to revolutionize regenerative and precision medicine. Genetically reprograming somatic cells to generate hiPSCs and genetic modification of hiPSCs are considered the key procedures for the study and application of hiPSCs. However, there are significant technical challenges for transgene delivery into somatic cells and hiPSCs since these cells are known to be difficult to transfect. The existing methods, such as viral transduction and chemical transfection, may introduce significant alternations to hiPSC culture which affect the potency, purity, consistency, safety, and functional capacity of hiPSCs. Therefore, generation and genetic modification of hiPSCs through non-viral approaches are necessary and desirable. Nanotechnology has revolutionized fields from astrophysics to biology over the past two decades. Increasingly, nanoparticles have been used in biomedicine as powerful tools for transgene and drug delivery, imaging, diagnostics, and therapeutics. The most successful example is the recent development of SARS-CoV-2 vaccines at warp speed to combat the 2019 coronavirus disease (COVID-19), which brought nanoparticles to the center stage of biomedicine and demonstrated the efficient nanoparticle-mediated transgene delivery into human body. Nanoparticles have the potential to facilitate the transgene delivery into the hiPSCs and offer a simple and robust approach. Nanoparticle-mediated transgene delivery has significant advantages over other methods, such as high efficiency, low cytotoxicity, biodegradability, low cost, directional and distal controllability, efficient in vivo applications, and lack of immune responses. Our recent study using magnetic nanoparticles for transfection of hiPSCs provided an example of the successful applications, supporting the potential roles of nanoparticles in hiPSC biology. This review discusses the principle, applications, and significance of nanoparticles in the transgene delivery to hiPSCs and their successful application in the development of COVID-19 vaccines.

Oncolytic Adenoviruses for Cancer Therapy

Many immuno-therapeutic strategies are currently being developed to fight cancer. In this scenario, oncolytic adenoviruses (Onc.Ads) have an interesting role for their peculiar tumor selectivity, safety, and transgene-delivery capability. The major strength of the Onc.Ads is the extraordinary immunogenicity that leads to a strong T-cell response, which, together with the possibility of the delivery of a therapeutic transgene, could be more effective than current strategies. In this review, we travel in the adenovirus (Ads) and Onc.Ads world, focusing on a variety of strategies that can enhance Onc.Ads antitumoral efficacy, passing through tumor microenvironment modulation. Onc.Ads-based therapeutic strategies constitute additional weapons in the fight against cancer and appear to potentiate conventional and immune checkpoint inhibitors (ICIs)-based therapies leading to a promising scenario.