Gene Therapy Strategies Research Articles

Gene Therapy: The Next-Generation Therapeutics and Their Delivery Approaches for Neurological Disorders.

Neurological conditions like neurodevelopmental disorders and neurodegenerative diseases are quite complex and often exceedingly difficult for patients. Most of these conditions are due to a mutation in a critical gene. There is no cure for the majority of these neurological conditions and the availability of disease-modifying therapeutics is quite rare. The lion’s share of the treatments that are available only provide symptomatic relief, as such, we are in desperate need of an effective therapeutic strategy for these conditions. Considering the current drug development landscape, gene therapy is giving us hope as one such effective therapeutic strategy. Consistent efforts have been made to develop gene therapy strategies using viral and non-viral vectors of gene delivery. Here, we have discussed both of these delivery methods and their properties. We have summarized the relative advantages and drawbacks of viral and non-viral vectors from the perspectives of safety, efficiency, and productivity. Recent developments such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene editing and its use in vivo have been described here as well. Given recent advancements, gene therapy shows great promise to emerge as a next-generation therapeutic for many of the neurodevelopmental and neurodegenerative conditions.

CRISPR/Cas9-Directed Gene Trap Constitutes a Selection System for Corrected BCR/ABL Leukemic Cells in CML.

Chronic myeloid leukaemia (CML) is a haematological neoplasm driven by the BCR/ABL fusion oncogene. The monogenic aspect of the disease and the feasibility of ex vivo therapies in haematological disorders make CML an excellent candidate for gene therapy strategies. The ability to abolish any coding sequence by CRISPR-Cas9 nucleases offers a powerful therapeutic opportunity to CML patients. However, a definitive cure can only be achieved when only CRISPR-edited cells are selected. A gene-trapping approach combined with CRISPR technology would be an ideal approach to ensure this. Here, we developed a CRISPR-Trap strategy that efficiently inserts a donor gene trap (SA-CMV-Venus) cassette into the BCR/ABL-specific fusion point in the CML K562 human cell line. The trapping cassette interrupts the oncogene coding sequence and expresses a reporter gene that enables the selection of edited cells. Quantitative mRNA expression analyses showed significantly higher level of expression of the BCR/Venus allele coupled with a drastically lower level of BCR/ABL expression in Venus+ cell fractions. Functional in vitro experiments showed cell proliferation arrest and apoptosis in selected Venus+ cells. Finally, xenograft experiments with the selected Venus+ cells showed a large reduction in tumour growth, thereby demonstrating a therapeutic benefit in vivo. This study represents proof of concept for the therapeutic potential of a CRISPR-Trap system as a novel strategy for gene elimination in haematological neoplasms.

Isogenic GAA-KO Murine Muscle Cell Lines Mimicking Severe Pompe Mutations as Preclinical Models for the Screening of Potential Gene Therapy Strategies.

Pompe disease (PD) is a rare disorder caused by mutations in the acid alpha-glucosidase (GAA) gene. Most gene therapies (GT) partially rely on the cross-correction of unmodified cells through the uptake of the GAA enzyme secreted by corrected cells. In the present study, we generated isogenic murine GAA-KO cell lines resembling severe mutations from Pompe patients. All of the generated GAA-KO cells lacked GAA activity and presented an increased autophagy and increased glycogen content by means of myotube differentiation as well as the downregulation of mannose 6-phosphate receptors (CI-MPRs), validating them as models for PD. Additionally, different chimeric murine GAA proteins (IFG, IFLG and 2G) were designed with the aim to improve their therapeutic activity. Phenotypic rescue analyses using lentiviral vectors point to IFG chimera as the best candidate in restoring GAA activity, normalising the autophagic marker p62 and surface levels of CI-MPRs. Interestingly, in vivo administration of liver-directed AAVs expressing the chimeras further confirmed the good behaviour of IFG, achieving cross-correction in heart tissue. In summary, we generated different isogenic murine muscle cell lines mimicking the severe PD phenotype, as well as validating their applicability as preclinical models in order to reduce animal experimentation.

THER-01. Precision brain tumor therapy by AAV-mediated oncogene editing

Pediatric high-grade glioma is a heterogeneous group of highly malignant tumors of the central nervous system, with a median overall survival of less than two years after diagnosis, demanding novel treatment options. One innovative approach is gene therapy, which has so far been hampered for cancer treatment owing to the lack of a system targeting tumor cells specifically. To overcome this limitation, we established a novel strategy for gene therapy, combining tumor cell-specific adeno-associated virus (AAV) variants with oncogene-specific CRISPR-Cas nucleases. We screened 177 different Cas9/gRNA combinations targeting the genes encoding H3K27M or BRAFV600E, and identified highly specific nucleases that edited the oncogenic allele but left the respective WT loci intact, which we validated by PCR amplicon sequencing. Next, we intravenously injected an AAV library engineered to encode its own capsid DNA into mice harboring patient-derived xenograft tumors driven by H3K27M or BRAFV600E. After 21 days, we resected neoplasms and separated mCherry-labeled tumor cells from normal surrounding cells by fluorescence-activated cell sorting. Using the DNA from tumor cells as template, we generated a second AAV library, which was utilized in another round of in vivo selection. At the end of each screen, DNA from tumor cells, surrounding cells, and control tissues (liver and spleen) was analyzed by amplicon sequencing. Strikingly, we identified multiple AAV variants that were highly and recurrently enriched in the analyzed tumor tissues. We are currently validating these variants by intravenously injecting selected, GFP-encoding AAVs to tumor-bearing mice and by subsequently analyzing their distribution throughout the aforementioned tissues. We will combine oncogene-specific nucleases with these validated AAV variants and analyze their anti-tumoral efficacy in a preclinical setting. Furthermore, we plan to adapt this approach to allografted mice, evaluating its feasibility and efficacy in syngeneic models.

Improving Control of Gene Therapy-Based Neurotrophin Delivery for Inner Ear Applications.

Background: Survival and integrity of the spiral ganglion is vital for hearing in background noise and for optimal functioning of cochlear implants. Numerous studies have demonstrated that supplementation of supraphysiologic levels of the neurotrophins BDNF and NT-3 by pumps or gene therapy strategies supports spiral ganglion survival. The endogenous physiological levels of growth factors within the inner ear, although difficult to determine, are likely extremely low within the normal inner ear. Thus, novel approaches for the long-term low-level delivery of neurotrophins may be advantageous. Objectives: This study aimed to evaluate the long-term effects of gene therapy-based low-level neurotrophin supplementation on spiral ganglion survival. Using an adenovirus serotype 28-derived adenovector delivery system, the herpes latency promoter, a weak, long expressing promoter system, has been used to deliver the BDNF or NTF3 genes to the inner ear after neomycin-induced ototoxic injury in mice. Results: Treatment of the adult mouse inner ear with neomycin resulted in acute and chronic changes in endogenous neurotrophic factor gene expression and led to a degeneration of spiral ganglion cells. Increased survival of spiral ganglion cells after adenoviral delivery of BDNF or NTF3 to the inner ear was observed. Expression of BDNF and NT-3 could be demonstrated in the damaged organ of Corti after gene delivery. Hearing loss due to overexpression of neurotrophins in the normal hearing ear was avoided when using this novel vector–promoter combination. Conclusion: Combining supporting cell-specific gene delivery via the adenovirus serotype 28 vector with a low-strength long expressing promoter potentially can provide long-term neurotrophin delivery to the damaged inner ear.

Identification of Hub Genes in Colorectal Adenocarcinoma by Integrated Bioinformatics.

An improved understanding of the molecular mechanism of colorectal adenocarcinoma is necessary to predict the prognosis and develop new target gene therapy strategies. This study aims to identify hub genes associated with colorectal adenocarcinoma and further analyze their prognostic significance. In this study, The Cancer Genome Atlas (TCGA) COAD-READ database and the gene expression profiles of GSE25070 from the Gene Expression Omnibus were collected to explore the differentially expressed genes between colorectal adenocarcinoma and normal tissues. The weighted gene co-expression network analysis (WGCNA) and differential expression analysis identified 82 differentially co-expressed genes in the collected datasets. Enrichment analysis was applied to explore the regulated signaling pathway in colorectal adenocarcinoma. In addition, 10 hub genes were identified in the protein–protein interaction (PPI) network by using the cytoHubba plug-in of Cytoscape, where five genes were further proven to be significantly related to the survival rate. Compared with normal tissues, the expressions of the five genes were both downregulated in the GSE110224 dataset. Subsequently, the expression of the five hub genes was confirmed by the Human Protein Atlas database. Finally, we used Cox regression analysis to identify genes associated with prognosis, and a 3-gene signature (CLCA1–CLCA4–GUCA2A) was constructed to predict the prognosis of patients with colorectal cancer. In conclusion, our study revealed that the five hub genes and CLCA1–CLCA4–GUCA2A signature are highly correlated with the development of colorectal adenocarcinoma and can serve as promising prognosis factors to predict the overall survival rate of patients.

Gene Therapy for Acquired and Genetic Cholestasis.

Cholestatic diseases can be caused by the dysfunction of transporters involved in hepatobiliary circulation. Although pharmacological treatments constitute the current standard of care for these diseases, none are curative, with liver transplantation being the only long-term solution for severe cholestasis, albeit with many disadvantages. Liver-directed gene therapy has shown promising results in clinical trials for genetic diseases, and it could constitute a potential new therapeutic approach for cholestatic diseases. Many preclinical gene therapy studies have shown positive results in animal models of both acquired and genetic cholestasis. The delivery of genes that reduce apoptosis or fibrosis or improve bile flow has shown therapeutic effects in rodents in which cholestasis was induced by drugs or bile duct ligation. Most studies targeting inherited cholestasis, such as progressive familial intrahepatic cholestasis (PFIC), have focused on supplementing a correct version of a mutated gene to the liver using viral or non-viral vectors in order to achieve expression of the therapeutic protein. These strategies have generated promising results in treating PFIC3 in mouse models of the disease. However, important challenges remain in translating this therapy to the clinic, as well as in developing gene therapy strategies for other types of acquired and genetic cholestasis.

Simple Autofluorescence-Restrictive Sorting of eGFP+ RPE Cells Allows Reliable Assessment of Targeted Retinal Gene Therapy

Gene therapy is a promising therapeutic modality for ocular diseases arising in and affecting the retina and choroid. In this context, delivering gene therapy to the multifunctional retinal pigment epithelium (RPE) cells situated between the retina and choroid is desired. Efficacy assessment of any gene therapy strategy, whether it is gene augmentation, inhibition, or editing is initially tested in vitro in cell models, where delivery is simple and efficient. However, efficacy assessment in vivo in animal models is far more complex and several factors can influence the result significantly. Here we report a simple fluorescence activated cell sorting (FACS)-based enrichment method for direct assessment of efficacy and potential off-target effects of gene therapy co-delivered with an eGFP reporter to murine RPE cells using subretinal administration. Isolation of true eGFP+ RPE cells by FACS is notoriously difficult due to their intrinsic autofluorescence resulting in decreased sensitivity and false positives. Combining retinal dissection and harvest of RPE cells with a FACS-gating strategy utilizing the GFP filter and a neighboring filter, to separate the eGFP signal from autofluorescence, allows a significant enrichment of gene therapy-targeted eGFP+ RPE cells. In our hands the method may provide quantitative and qualitative advances in terms of up to 7-fold enrichment of true eGFP+ RPE cells compared to a standard protocol. The isolated cells can subsequently be utilized for reliable assessment of changes in DNA, RNA, or protein. This method allows proof-of-principle analysis of early gene therapy development and investigation of new delivery strategies or therapeutic approaches targeting RPE cells in vivo.

Optimized lentiviral vector to restore full-length dystrophin via a cell-mediated approach in a mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the DMD gene. Restoration of full-length dystrophin protein in skeletal muscle would have therapeutic benefit, but lentivirally mediated delivery of such a large gene in vivo has been hindered by lack of tissue specificity, limited transduction, and insufficient transgene expression. To address these problems, we developed a lentiviral vector, which contains a muscle-specific promoter and sequence-optimized full-length dystrophin, to constrain dystrophin expression to differentiated myotubes/myofibers and enhance the transgene expression. We further explored the efficiency of restoration of full-length dystrophin in vivo, by grafting DMD myoblasts that had been corrected by this optimized lentiviral vector intramuscularly into an immunodeficient DMD mouse model. We show that these lentivirally corrected DMD myoblasts effectively reconstituted full-length dystrophin expression in 93.58% ± 2.17% of the myotubes in vitro. Moreover, dystrophin was restored in 64.4% ± 2.87% of the donor-derived regenerated muscle fibers in vivo, which were able to recruit members of the dystrophin-glycoprotein complex at the sarcolemma. This study represents a significant advance over existing cell-mediated gene therapy strategies for DMD that aim to restore full-length dystrophin expression in skeletal muscle.

Blood-brain barrier-penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy.

We designed a unique nanocapsule for efficient single CRISPR-Cas9 capsuling, noninvasive brain delivery and tumor cell targeting, demonstrating an effective and safe strategy for glioblastoma gene therapy. Our CRISPR-Cas9 nanocapsules can be simply fabricated by encapsulating the single Cas9/sgRNA complex within a glutathione-sensitive polymer shell incorporating a dual-action ligand that facilitates BBB penetration, tumor cell targeting, and Cas9/sgRNA selective release. Our encapsulating nanocapsules evidenced promising glioblastoma tissue targeting that led to high PLK1 gene editing efficiency in a brain tumor (up to 38.1%) with negligible (less than 0.5%) off-target gene editing in high-risk tissues. Treatment with nanocapsules extended median survival time (68 days versus 24 days in nonfunctional sgRNA-treated mice). Our new CRISPR-Cas9 delivery system thus addresses various delivery challenges to demonstrate safe and tumor-specific delivery of gene editing Cas9 ribonucleoprotein for improved glioblastoma treatment that may potentially be therapeutically useful in other brain diseases.

Principles of translational gene therapy for neuromuscular diseases

In recent years the theoretical hope has become reality and the first hereditary neuromuscular diseases have become causally treatable. Neuromuscular diseases have thus become the pacemaker of this form of therapy for the whole of neurology. This article describes the principles of precision gene therapy for neurogenetic diseases using examples of neuromuscular diseases. Various strategies of gene therapy have become established and are being tested in preclinical and clinical trials and evaluated as approved forms for long-term efficacy. The aim of every gene therapy is the modification or introduction of the target gene with initiation of adegradation of dysfunctional proteins. Various techniques, such as gene transfer, gene substitution or gene editing in vivo and ex vivo are now usable. For example, amodification of the pre-mRNA using antisense oligonucleotides or RNA interference (siRNA) can be used for exon skipping. An initiation of gene expression to produce the target protein can be based on amodification of the DNA by means of gene replacement, cell-based therapy (iPS cells), regulation by compensatory proteins or pharmacological treatment with so-called small molecules. Each method has advantages and complex disadvantages that must be individually evaluated. Phenotypic peculiarities of arare disease often only become apparent through specific translational therapy. It is already becoming obvious that avery early point in timing of gene therapy is probably the most effective. Newborn screening is therefore gaining additional importance as early diagnosis can achieve the best possible success of therapies, possibly even preventively.

Emerging drugs for the treatment of hereditary angioedema due to C1-inhibitor deficiency

ABSTRACT Introduction Hereditary angioedema due to C1-inhibitor (C1-INH-HAE) is a rare disease characterized by unpredictable swelling attacks that may be life-threatening when affecting the upper airways. Understanding the pathophysiology of HAE and the mechanism of bradykinin-mediated angioedema allowed the development of new therapies for the treatment of HAE: clinical trials are ongoing to expand the number of drugs available for on-demand treatment and prophylaxis. Areas covered Authors discuss the products that have been used to treat this disease for many years and present the most recently marketed products and those which are under development. Expert opinion Significant therapeutic progress has been made in HAE. In particular, drugs targeting specific molecules involved in the angioedema formation were developed and studies with new drugs are ongoing. In the coming years, more effective therapies with easier administration route options for on-demand treatment and long-term prophylaxis will be available to treat this disease and the variety of patients. Gene therapy strategies may offer a definitive treatment. High costs of current and new drugs may be a limiting factor for their availability, especially in developing countries.

Insulin receptor activation by proinsulin preserves synapses and vision in retinitis pigmentosa

Synaptic loss, neuronal death, and circuit remodeling are common features of central nervous system neurodegenerative disorders. Retinitis pigmentosa (RP), the leading cause of inherited blindness, is a group of retinal dystrophies characterized by photoreceptor dysfunction and death. The insulin receptor, a key controller of metabolism, also regulates neuronal survival and synaptic formation, maintenance, and activity. Indeed, deficient insulin receptor signaling has been implicated in several brain neurodegenerative pathologies. We present evidence linking impaired insulin receptor signaling with RP. We describe a selective decrease in the levels of the insulin receptor and its downstream effector phospho-S6 in retinal horizontal cell terminals in the rd10 mouse model of RP, as well as aberrant synapses between rod photoreceptors and the postsynaptic terminals of horizontal and bipolar cells. A gene therapy strategy to induce sustained proinsulin, the insulin precursor, production restored retinal insulin receptor signaling, by increasing S6 phosphorylation, without peripheral metabolic consequences. Moreover, proinsulin preserved photoreceptor synaptic connectivity and prolonged visual function in electroretinogram and optomotor tests. These findings point to a disease-modifying role of insulin receptor and support the therapeutic potential of proinsulin in retinitis pigmentosa.

Engineering silica-polymer hybrid nanosystems for dual drug and gene delivery.

In recent years, it has been shown that a combination of different antitumour strategies involving distinct therapeutic agents, such as chemical compounds and genetic material, could result in an effective therapeutic activity that is much higher than that obtained by conventionally used individual approaches. Therefore, the main goal of this work was to develop a new hybrid nanosystem based on mesoporous silica nanoparticles and polymers to efficiently transport and deliver drug and plasmid DNA into cancer cells. Moreover, its potential to mediate a combinatorial antitumour strategy involving epirubicin and herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy was evaluated. For this purpose, various cationic polymers were assessed, including poly(β-amino ester) homopolymer, gelatine type A, gelatine type B, and poly(ethylene glycol)-b-poly(2-aminoethyl methacrylate hydrochloride) block copolymer. The obtained results show that using different polymers leads to nanosystems with different physicochemical properties and, consequently, different biological activities. The best formulation was obtained for hybrid nanosystems coated with PEG-b-PAMA. They demonstrated the ability to cotransport and codeliver an anticancer drug and plasmid DNA and effectively mediate the combined antitumour strategy in 2D and 3D tumour cell culture models. In summary, we developed a novel silica- and polymer-based nanosystem able to mediate a dual chemotherapeutic and suicide gene therapy strategy with a much higher therapeutic effect than that obtained through the use of individual approaches, showing its potential for cancer treatment.

A Single Transcript Knockdown-Replacement Strategy Employing 5' UTR Secondary Structures to Precisely Titrate Rescue Protein Translation.

One overarching goal of gene therapy is the replacement of faulty genes with functional ones. A significant hurdle is presented by the fact that under- or over-expression of a protein may cause disease as readily as coding mutations. There is a clear and present need for pipelines to translate experimentally validated gene therapy strategies to clinical application. To address this we developed a modular, single-transgene expression system for replacing target genes with physiologically expressed variants. In order to accomplish this, we first designed a range of 5’ UTR “attenuator” sequences which predictably diminish translation of the paired gene. These sequences provide wide general utility by allowing control over translation from high expression, ubiquitous promoters. Importantly, we demonstrate that this permits an entirely novel knockdown and rescue application by pairing microRNA-adapted shRNAs alongside their respective replacement gene on a single transcript. A noteworthy candidate for this corrective approach is the degenerative and uniformly fatal motor neuron disease ALS. A strong proportion of non-idiopathic ALS cases are caused by varied mutations to the SOD1 gene, and as clinical trials to treat ALS are being initiated, it is important to consider that loss-of-function mechanisms contribute to its pathology as strongly as any other factor. As a generalized approach to treat monogenic diseases caused by heterogeneous mutations, we demonstrate complete and predictable control over replacement of SOD1 in stable cell lines by varying the strength of attenuators.

Engineering stem cells to produce exosomes with enhanced bone regeneration effects: an alternative strategy for gene therapy

BackgroundExosomes derived from stem cells have been widely studied for promoting regeneration and reconstruction of multiple tissues as “cell-free” therapies. However, the applications of exosomes have been hindered by limited sources and insufficient therapeutic potency.ResultsIn this study, a stem cell-mediated gene therapy strategy is developed in which mediator mesenchymal stem cells are genetically engineered by bone morphogenetic protein-2 gene to produce exosomes (MSC-BMP2-Exo) with enhanced bone regeneration potency. This effect is attributed to the synergistic effect of the content derived from MSCs and the up-regulated BMP2 gene expression. The MSC-BMP2-Exo also present homing ability to the injured site. The toxic effect of genetical transfection vehicles is borne by mediator MSCs, while the produced exosomes exhibit excellent biocompatibility. In addition, by plasmid tracking, it is interesting to find a portion of plasmid DNA can be encapsulated by exosomes and delivered to recipient cells.ConclusionsIn this strategy, engineered MSCs function as cellular factories, which effectively produce exosomes with designed and enhanced therapeutic effects. The accelerating effect in bone healing and the good biocompatibility suggest the potential clinical application of this strategy.Graphical

Oncolytic therapy with vaccinia virus carrying IL-24 for hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a highly refractory cancer associated with increasing mortality, which currently lacks effective treatment options. Interleukin-24 (IL-24) is a novel tumor suppressor cytokine that can selectively induce cancer cell apoptosis, and it has been utilized as a cancer gene therapy strategy. The vaccinia virus is a promising strategy for cancer therapy, owing to its direct viral lytic effects, as well as a vehicle to overexpress therapeutic transgenes.MethodsWe constructed a recombinant oncolytic vaccinia viruse (VG9-IL-24) based on vaccinia virus Guang9 (VG9) harboring the IL-24 gene. In vitro, we assessed the replication of VG9-IL-24 in HCC cell lines and normal liver cells and evaluated the cytotoxicity in different cell lines; then, we determined the expression of IL-24 by RT-PCR and ELISA. We examined apoptosis and cell cycle progression in SMMC-7721 cells treated with VG9-IL-24 by flow cytometry. In vivo, we established the SMMC-7721 xenograft mouse model to evaluate the antitumor effects of VG9-IL-24.ResultsIn vitro, VG9-IL-24 efficiently infected HCC cell lines, but not normal liver cells, and resulted in a high level of IL-24 expression and significant cytotoxicity. Moreover, VG9-IL-24 induced an increase in the proportion of apoptotic cells and blocked the SMMC-7721 cell cycle in the G2/M phase. In vivo, tumor growth was significantly suppressed and the survival was prolonged in VG9-IL-24-treated mice.ConclusionsVaccinia virus VG9-mediated gene therapy might be an innovative treatment for cancer with tumor-specific lysis and apoptosis-inducing effects. VG9-IL-24 exhibited enhanced antitumor effects and is a promising candidate for HCC therapy.

Site-Specific Integration of TRAIL in iPSC-Derived Mesenchymal Stem Cells for Targeted Cancer Therapy.

Mesenchymal stem cells (MSCs) are a promising cellular vehicle for transferring anti-cancer factors to malignant tumors. Currently, a variety of anti-cancer agents, including the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), have been loaded into MSCs derived from a range of sources through different engineering methods. These engineered MSCs exhibit enormous therapeutic potential for various cancers. To avoid the intrinsic defects of MSCs derived from tissues and the potential risk of viral vectors, TRAIL was site-specifically integrated into the ribosomal DNA (rDNA) locus of human-induced pluripotent stem cells (iPSCs) using a non-viral rDNA-targeting vector and transcription activator-like effector nickases (TALENickases). These genetically modified human iPSCs were differentiated into an unlimited number of homogeneous induced MSCs (TRAIL-iMSCs) that overexpressed TRAIL in both culture supernatants and cell lysates while maintaining MSC-like characteristics over continuous passages. We found that TRAIL-iMSCs significantly induced apoptosis in A375, A549, HepG2, and MCF-7 cells in vitro. After intravenous infusion, TRAIL-iMSCs had a prominent tissue tropism for A549 or MCF-7 xenografts and significantly inhibited tumor growth through the activation of apoptotic signaling pathways without obvious side effects in tumor-bearing mice models. Altogether, our results showed that TRAIL-iMSCs have strong anti-tumor effects in vitro and in vivo on a range of cancers. This study allows for the development of an unlimited number of therapeutic gene-targeted MSCs with stable quality and high homogeneity for cancer therapy, thus highlighting a universal and safe strategy for stem cell-based gene therapy with high potential for clinical applications.

Gene therapy of yeast NDI1 on mitochondrial complex I dysfunction in rotenone-induced Parkinson\u2019s disease models in vitro and vivo

PurposeParkinson's disease (PD) is the second most common neurodegenerative disease without cure or effective treatment. This study explores whether the yeast internal NADH-quinone oxidoreductase (NDI1) can functionally replace the defective mammalian mitochondrial complex I, which may provide a gene therapy strategy for treating sporadic PD caused by mitochondrial complex I dysfunction.MethodRecombinant lentivirus expressing NDI1 was transduced into SH-SY5Y cells, or recombinant adeno-associated virus type 5 expressing NDI1 was transduced into the right substantia nigra pars compacta (SNpc) of mouse. PD cell and mouse models were established by rotenone treatment. The therapeutic effects of NDI1 on rotenone-induced PD models in vitro and vivo were assessed in neurobehavior, neuropathology, and mitochondrial functions, by using the apomorphine-induced rotation test, immunohistochemistry, immunofluorescence, western blot, complex I enzyme activity determination, oxygen consumption detection, ATP content determination and ROS measurement.ResultsNDI1 was expressed and localized in mitochondria in SH-SY5Y cells. NDI1 resisted rotenone-induced changes in cell morphology, loss of cell viability, accumulation of α-synuclein and pS129 α-synuclein, mitochondrial ROS production and mitochondria-mediated apoptosis. The basal and maximal oxygen consumption, mitochondrial coupling efficiency, basal and oligomycin-sensitive ATP and complex I activity in cell model were significantly increased in rotenone + NDI1 group compared to rotenone + vector group. NDI1 was efficiently expressed in dopaminergic neurons in the right SNpc without obvious adverse effects. The rotation number to the right side (NDI1-treated side) was significantly increased compared to that to the left side (untreated side) in mouse model. The number of viable dopaminergic neurons, the expression of tyrosine hydroxylase, total and maximal oxygen consumption, mitochondrial coupling efficiency and complex I enzyme activity in right substantia nigra, and the content of dopamine in right striatum were significantly increased in rotenone + NDI1 group compared to rotenone + vector group.ConclusionYeast NDI1 can rescue the defect of oxidative phosphorylation in rotenone-induced PD cell and mouse models, and ameliorate neurobehavioral and neuropathological damages. The results may provide a basis for the yeast NDI1 gene therapy of sporadic PD caused by mitochondrial complex I dysfunction.

Sonoporation based on repeated vaporization of gold nanodroplets.

BackgroundGold nanodroplets (AuNDs) have been proposed as agents for photothermal therapy and photoacoustic imaging. Previously, we demonstrated that the sonoporation can be more effectively achieved with synchronized optical and acoustic droplet vaporization. By applying a laser pulse at the rarefactional phase of the ultrasound (US) pulse, the vaporization threshold can be reached at a considerably lower laser average power. However, a large loading quantity of the AuNDs may increase the risk of air embolism. The destruction of phase‐shifted AuNDs at the inertial cavitation stage leads to a reduced drug delivery performance. And it also causes instability of echogenicity during therapeutic monitoring.PurposeIn this study, we propose to further improve the sonoporation effectiveness with repeated vaporization. In other words, the AuNDs repeatedly undergo vaporization and recondensation so that sonoporation effects are accumulated over time at lower energy requirements. Previously, repeated vaporization has been demonstrated as an imaging contrast agent. In this study, we aim to adopt this repeated vaporization scheme for sonoporation.MethodsPerfluoropentane NDs with a shell made of human serum albumin were used as the US contrast agents. Laser pulses at 808 nm and US pulses of 1 MHz were delivered for triggering vaporization and inertial cavitation of NDs. We detected the vaporization and cavitation effects under different activation firings, US peak negative pressures (PNPs), and laser fluences using 5‐ and 10‐MHz focused US receivers. Numbers of calcein‐AM and propidium iodide signals uptake by BNL hepatocarcinoma cancer cells were used to evaluate the sonoporation and cell death rate of the cells.ResultsWe demonstrate that sonoporation can be realized based on repeatable vaporization instead of the commonly adopted inertial cavitation effects. In addition, it is found that the laser fluence and the acoustic pressure can be reduced. As an example, we demonstrate that the acoustic and optical energy for achieving a similar level of sonoporation rate can be as low as 0.44 MPa for the US PNP and 4.01 mJ/cm2 for the laser fluence, which are lower than those with our previous approach (0.53 MPa and 4.95 mJ/cm2, respectively).ConclusionWe demonstrated the feasibility of vaporization‐based sonoporation at a lower optical and acoustic energy. It is an advantageous method that can enhance drug delivery efficiency, therapeutic safety and potentially deliver an upgraded gene therapy strategy for improved theragnosis.