Chemical Epigenetic Regulation of Adeno-Associated Virus Delivered Transgenes.

Adeno-associated virus (AAV) is a powerful gene therapy vector that has been used in several FDA-approved therapies as well as in multiple clinical trials. This vector has high therapeutic versatility with the ability to deliver genetic payloads to a variety of human tissue types, yet there is currently a lack of transgene expression control once the virus is administered. There are also times when transgene expression is too low for the desired therapeutic outcome, necessitating high viral dose administration resulting in possible immunological complications. Herein, we validate a chemically controllable AAV transgene expression technology in vitro that utilizes bifunctional molecules known as chemical epigenetic modifiers (CEMs). These compounds employ endogenous epigenetic machinery to specifically enhance transgene expression of episomal DNA. A recombinant AAV (rAAV) was designed to both deliver the reporter transgene as well as deliver a synthetic zinc finger (ZFs) protein fused to FK506 binding protein (FKBP). These synthetic ZFs target a DNA-binding array sequence upstream of the promoter expressing the AAV transgene to specifically enhance AAV transgene expression in the presence of a CEM. The transcriptional activating compound CEM87 functions by recruiting the epigenetic transcription activator bromodomain-containing protein 4 (BRD4), increasing AAV transgene activity up to fivefold in a dose-dependent manner in HEK293T cells. The highest levels of transgene product activity are seen 24 h following CEM87 treatment. Additionally, the CEM87-mediated enhancement of different transgene products with either Luciferase or green fluorescent protein (GFP) was observed in multiple cell lines and enhancement of transgene expression was capsid serotype independent. The impact of CEM87 activity can be disrupted through drug removal or chemical recruitment site competition with FK506, thus demonstrating the reversibility of the impact of CEM87 on transgene expression. Collectively, this chemically controllable rAAV transgene technology provides temporal gene expression control that could increase the safety and efficiency of AAV-based research and therapies.

AAV-mediated expression of HLA-G for the prevention of experimental ocular graft vs. host disease.

Ocular graft versus host disease (OGvHD) develops after allogeneic hematopoietic stem cell transplantation (HSCT) and manifests as ocular surface inflammatory disease. This study evaluated the efficacy of adeno-associated virus (AAV) gene therapy encoding human leukocyte antigen G (HLA-G) to inhibit OGvHD. A major histocompatibility mismatch chronic OGvHD murine model was evaluated. 7 days after HSCT, mice were dosed subconjunctivally with scAAV8-HLA-G1/5 (1 x 109 vg/eye), topical cyclosporine (twice daily), or left untreated. Body weights and tear production (red thread test) were recorded, and eyelid, corneal opacity, and corneal fluorescein retention were scored through day 44 after HSCT. Tissues were collected for vector biodistribution, ocular histology, and immunofluorescence. Compared with untreated HSCT eyes, those dosed with scAAV8-HLA-G1/5 had significantly reduced clinical inflammatory signs of OGvHD. On histology, eyes that received scAAV8-HLA-G1/5 or cyclosporine had a significantly lower mean limbal mononuclear cell count when compared with non-treated HSCT eyes. HLA-G immunofluorescence was detected in the subconjunctiva and peripheral cornea in HSCT animals treated with scAAV8-HLA-G1/5. Vector genomes were detected in the lacrimal gland, but not in the other tested organs. These results provide evidence that subconjunctival AAV targets ocular surface and corneal disease and support that HLA-G-based gene therapy may be an effective treatment for OGvHD.

Human Hepatocyte Transduction with Adeno-Associated Virus Vector.

As the adeno-associated virus (AAV) vectors hold unique advantages over other viral vectors, AAV gene therapy has accumulated rapid progress and development. Liver-targeted gene therapy by AAV vectors has been successfully applied in clinical trials for many diseases. Low transduction efficiency and high prevalence of neutralizing antibodies (Nabs), however, are the major obstacles to further translate this therapeutic strategy into clinical trials. Pre-clinical evaluation on hepatocytes could help to elucidate the tropism of AAV serotypes for liver-targeted gene therapy, and could also provide a test model to develop novel AAV mutants with Nabs evasion and high liver tropism. Here, we described the basic laboratory procedure to apply the AAV vector to transduce human hepatocytes in vitro and in vivo with some tips gained from our own experience.

Inhibition of experimental autoimmune uveitis by intravitreal AAV-Equine-IL10 gene therapy.

Equine recurrent uveitis (ERU) is a spontaneous, painful, and vision threatening disease affecting up to 25% of equine populations worldwide. Current treatments of ERU are non-specific and have many side effects which limits them to short-term use. In order to develop an effective therapy for ERU, we investigated the use of adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by equine interleukin-10 (Equine-IL10). The purpose of this study was to evaluate the therapeutic efficacy of a single intravitreal (IVT) dose of AAV8-Equine-IL10 gene therapy for inhibition of experimental autoimmune uveitis (EAU) in rats. Each rat was dosed intravitreally (IVT) in both eyes with either balanced salt solution (BSS) (control; n = 4), AAV8-Equine-IL10 at a low dose (2.4x109 vg; n = 5) or high dose (2.4x1010 vg; n = 5). EAU was induced in all groups of rats 7 days after IVT injections and euthanized 21 days post-injection. Ophthalmic examination and aqueous humor (AH) cell counts were recorded with the observer blinded to the treatment groups. Histopathology and qPCR were performed on selected ocular tissues. Data presented herein demonstrate that AAV8-Equine-IL10 treated rats exhibited a significant decrease in clinical inflammatory scores and AH cell counts compared to BSS-treated EAU eyes on days 10, 12 and 14 post EAU induction at both administered vector doses. Mean cellular histologic infiltrative scores were also significantly less in AAV8-Equine-IL10 dosed rats compared to the BSS group. Intravitreal injection of AAV8-Equine-IL10 resulted in Equine-IL10 cDNA expression in the ciliary body, retina, cornea, and optic nerve in a dose-dependent manner. A single IVT injection of AAV8-Equine-IL10 appeared to be well-tolerated and inhibited EAU even at the lowest administered dose. These results demonstrate safety and efficacy of AAV8-Equine-IL10 to prevent EAU and support continued exploration of AAV gene therapy for the treatment of equine and perhaps human recurrent uveitis.

A Gambogic Acid-Loaded Delivery System Mediated by Ultrasound-Targeted Microbubble Destruction: A Promising Therapy Method for Malignant Cerebral Glioma.

Background: The blood-brain barrier (BBB) inhibits the delivery of macromolecular chemotherapeutic drugs to brain tumors, leading to low utilization rates and toxic side effects to surrounding tissues and organs. Ultrasonic targeted microbubble destruction (UTMD) technology can open the BBB, leading to a new type of drug delivery system with particular utility in glioma. Purpose: We have developed a new type of drug-loaded microbubble complex based on poly(lactic-co-glycolic acid) (PLGA) that targets gambogic acid (GA) to the area of brain tumors through UTMD. Methods: GA/PLGA nanoparticles were prepared by the double emulsification method, and cationic microbubbles (CMBs) were prepared by a thin film hydration method. The GA/PLGA-CMB microbubble complex was assembled through electrostatic attractions and was characterized chemically. The anti-glioblastoma effect of GA/PLGA-CMB combined with focused ultrasound (FUS) was evaluated by biochemical and imaging assays in cultured cells and model mice. Results: GA/PLGA-CMB combined with FUS demonstrated a significant inhibitory effect on glioblastoma cell lines U87 and U251 as compared with controls (P<0.05). Tumor access and imaging analyses demonstrated that administration of GA/PLGA-CMBs combined with FUS can open the BBB and target the treatment of glioblastoma in a mouse model, as compared with control groups (P<0.05). Conclusion: The combination of PLGA-CMB with FUS provides an effective and biocompatible drug delivery system, and its application to the delivery of GA in a mouse glioblastoma model was successful.

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models.

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SC) injections offer advantages over other intraocular administration routes as they are simple, safe, require only local anesthesia, and are usually performed in an outpatient setting. Although SC injections in small animals usually require the assistance of an operating microscope due to the size of the eye, they are widely utilized for drug delivery, including gene therapy vectors. Previous work has demonstrated that SC injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases. Herein, a detailed protocol is presented for SC injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

Maternal tobacco exposure during pregnancy and allergic rhinitis in offspring: A systematic review and meta-analysis.

BACKGROUND: Maternal tobacco exposure during pregnancy is known to cause a potential hazard to the offspring's health. So far, published studies have shown no consistent results with whether tobacco exposure in utero is causally linked to the development of allergic rhinitis in offspring. The aim of this study was to comprehensively evaluate the association between maternal tobacco exposure during pregnancy and allergic rhinitis in offspring by meta-analysis and to provide reference for clinical work. METHODS: Literatures were searched in CNKI, Wanfang Data, VIP, SinoMed, PubMed, Web of science and Embase up to September 30,2020. Screening, inclusion, quality assessment, data extraction and data analysis of the literatures were conducted. Meta-analysis was performed with Revman 5.3 and State15.1 software. Odds ratio (OR) and 95%CI were used as observation indicators. RESULTS: We had retrieved 16 articles with 22 independent datasets and 11,49,879 sample size. When all the studies were analyzed together, the results showed that maternal smoking exposure during pregnancy would increase the risk of allergic rhinitis in offspring (OR = 1.13, 95%CI:1.02-1.26), especially maternal passive smoking during pregnancy (OR = 1.39, 95%CI:1.05-1.84). But subgroup analysis showed that maternal active smoking during pregnancy was only significantly associated with offspring allergic rhinitis in cross-sectional studies (OR = 1.24, 95%CI:1.07-1.45) and study done in America study (OR = 1.22, 95%CI:1.05-1.42). CONCLUSIONS: Tobacco exposure during pregnancy could increase the risk of allergic rhinitis in offspring. The importance of avoiding prenatal tobacco exposure should be emphasized more for the health of next generation in the public.

Harnessing the Natural Biology of Adeno-Associated Virus to Enhance the Efficacy of Cancer Gene Therapy.

Adeno-associated virus (AAV) was first characterized as small "defective" contaminant particles in a simian adenovirus preparation in 1965. Since then, a recombinant platform of AAV (rAAV) has become one of the leading candidates for gene therapy applications resulting in two FDA-approved treatments for rare monogenic diseases and many more currently in various phases of the pharmaceutical development pipeline. Herein, we summarize rAAV approaches for the treatment of diverse types of cancers and highlight the natural anti-oncogenic effects of wild-type AAV (wtAAV), including interactions with the cellular host machinery, that are of relevance to enhance current treatment strategies for cancer.

Adeno-Associated Virus Mediated Gene Therapy for Corneal Diseases.

According to the World Health Organization, corneal diseases are the fourth leading cause of blindness worldwide accounting for 5.1% of all ocular deficiencies. Current therapies for corneal diseases, which include eye drops, oral medications, corrective surgeries, and corneal transplantation are largely inadequate, have undesirable side effects including blindness, and can require life-long applications. Adeno-associated virus (AAV) mediated gene therapy is an optimistic strategy that involves the delivery of genetic material to target human diseases through gene augmentation, gene deletion, and/or gene editing. With two therapies already approved by the United States Food and Drug Administration and 200 ongoing clinical trials, recombinant AAV (rAAV) has emerged as the in vivo viral vector-of-choice to deliver genetic material to target human diseases. Likewise, the relative ease of applications through targeted delivery and its compartmental nature makes the cornea an enticing tissue for AAV mediated gene therapy applications. This current review seeks to summarize the development of AAV gene therapy, highlight preclinical efficacy studies, and discuss potential applications and challenges of this technology for targeting corneal diseases.

Adeno-Associated Virus Vector Mobilization, Risk Versus Reality.

Recombinant adeno-associated viral (rAAV) vector mobilization is a largely theoretical process in which intact AAV vectors spread or "mobilize" from transduced cells and infect additional cells within, or external of, the initial host. This process can be helper virus-independent (vector alone) or helper virus-dependent (de novo rAAV production facilitated by superinfection of both wild-type AAV [wtAAV] and Adenovirus 5 [Ad] helper virus). Herein, rAAV production and mobilization with and without wtAAV were analyzed following plasmid transfection or viral transduction utilizing well-established in vitro conditions and analytical measurements. During in vitro production, wtAAV produced the highest titer with rAAV-luc (4.1 kb), rAAV-IDUA (3.7 kb), and rAAV-Nano-dysferlin (4.9 kb) generating 2.5-, 5.9-, or 10.7-fold lower amounts, respectively. Surprisingly, cotransfection of a wtAAV and an rAAV plasmid resulted in a uniform decrease in production of wtAAV in all instances with a concomitant increase of rAAV such that wtAAV:rAAV titers were at a ratio of 1:1 for all constructs investigated. These results were shown to be independent of the rAAV transgenic sequence, size, transgene, or promoter choice and point to novel aspects of wtAAV complementation that enhance current vector production systems yet to be defined. In a mobilization assay, a sizeable amount of rAAV recovered from infected 293 cell lysate remained intact and competent for a secondary round of infection (termed Ad-independent mobilization). In rAAV-infected cells coinfected with Ad and wtAAV, rAAV particle production was increased >50-fold compared with no Ad conditions. In addition, Ad-dependent rAAV vectors mobilized and resulted in >1,000-fold transduction upon a subsequent second-round infection, highlighting the reality of these theoretical safety concerns that can be manifested under various conditions. Overall, these studies document and signify the need for mobilization-resistant vectors and the opportunity to derive better vector production systems.

Ocular Tolerability and Immune Response to Corneal Intrastromal AAV-IDUA Gene Therapy in New Zealand White Rabbits.

The chronic ocular toxicity, tolerability, and inflammation following corneal intrastromal injection of saline or escalating doses of an adeno-associated virus (AAV) containing a codon-optimized α-l-iduronidase (AAV-opt-IDUA) expression cassette were evaluated in New Zealand White rabbits. Corneal opacity following corneal intrastromal injection resolved by 24 h. Mild elevation of clinical ocular inflammation was observed 24 h after injection, but it returned to baseline by day 7 and no abnormalities were noted through 6 months of observation after injection. Vector genomes and IDUA cDNA were detected in the injected corneas in a dose-dependent manner. Both the lowest administered AAV-opt-IDUA dose, shown to be effective in mucopolysaccharidosis type I (MPS I) dogs, and a 10-fold higher dose of AAV-opt-IDUA resulted in no detectable immunologic response or adverse effect in rabbits. Vector genomes outside of the eye were rarely detected following corneal intrastromal injection of AAV-opt-IDUA, and neutralizing antibodies to the AAV capsid were not present at the experimental conclusion. This study, combined with our previous studies in MPS I dogs, suggests that AAV-opt-IDUA corneal gene therapy following corneal intrastromal injection of AAV-opt-IDUA has the potential to prevent and reverse blindness in MPS I patients in a safe and effective manner.

Preparation and Administration of Adeno-associated Virus Vectors for Corneal Gene Delivery.

Gene delivery approaches using adeno-associated virus (AAV) vectors are currently the preferred method for human gene therapy applications and have demonstrated success in clinical trials for a diverse set of diseases including retinal blindness. To date, no clinical trials using AAV gene therapy in the anterior eye have been initiated; however, corneal gene delivery appears to be an attractive approach for treating both corneal and ocular surface diseases. Multiple preclinical studies by our lab and others have demonstrated efficient AAV vector-mediated gene delivery to the cornea for immunomodulation, anti-vascularization, and enzyme supplementation. Interestingly, the route of AAV vector administration and nuances such as administered volume influence vector tropism and transduction efficiency. In this chapter, a detailed protocol for AAV vector production and specific approaches for AAV-mediated gene transfer to the cornea via subconjunctival and intrastromal injections are described.

Intrastromal Gene Therapy Prevents and Reverses Advanced Corneal Clouding in a Canine Model of Mucopolysaccharidosis I.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disease characterized by severe phenotypes, including corneal clouding. MPS I is caused by mutations in alpha-l-iduronidase (IDUA), a ubiquitous enzyme that catalyzes the hydrolysis of glycosaminoglycans. Currently, no treatment exists to address MPS I corneal clouding other than corneal transplantation, which is complicated by a high risk for rejection. Investigation of an adeno-associated virus (AAV) IDUA gene addition strategy targeting the corneal stroma addresses this deficiency. In MPS I canines with early or advanced corneal disease, a single intrastromal AAV8G9-IDUA injection was well tolerated at all administered doses. The eyes with advanced disease demonstrated resolution of corneal clouding as early as 1 week post-injection, followed by sustained corneal transparency until the experimental endpoint of 25 weeks. AAV8G9-IDUA injection in the MPS I canine eye with early corneal disease prevented the development of advanced corneal changes while restoring clarity. Biodistribution studies demonstrated vector genomes in ocular compartments other than the cornea and in some systemic organs; however, a capsid antibody response was detected in only the highest dosed subject. Collectively, the results suggest that intrastromal AAV8G9-IDUA therapy prevents and reverses visual impairment associated with MPS I corneal clouding.

A Fixed-Depth Microneedle Enhances Reproducibility and Safety for Corneal Gene Therapy.

PURPOSE: Drug delivery directly to the corneal stroma currently relies on microscopic injections that demonstrate low reproducibility and clinician-dependent variability. With use of biological drugs such as adeno-associated viral (AAV) vectors, precise and consistent drug deposition is critical to reduce concerns related to off-target transduction and the host's immune response to the viral capsid and/or transgene-derived product. Therefore, a precise corneal injection (PCI) microneedle was designed to allow accurate depth-specific injections into the corneal stroma in a macroscopic setting. METHODS: High-frequency ultrasound and confocal microscopy demonstrated the consistent ability to predetermine the precise injection depth using PCI needles of varying sizes. Next, a comparison between a standard 31-G needle and PCI needles was performed in vivo using AAV vector gene delivery. RESULTS: Intrastromal corneal injections using the PCI microneedle resulted in less vector leakage at the site of injection and fewer anterior chamber penetrations compared with a standard 31-G needle. Although reporter gene expression appeared similar when the vector was administered with either needle type, a trend toward increased vector genomes was noted in the PCI-injected corneas at the experimental conclusion. As hypothesized, corneal perforation resulted in increased detection of AAV vector genomes in nontarget tissues, highlighting the importance of consistency for biological drug applications in the cornea. CONCLUSIONS: Further development of the PCI microneedle is warranted especially for AAV corneal gene therapy and offers the potential to enhance transduction while significantly reducing safety concerns and intraclinician and interclinician injection variability.

AAV-mediated expression of HLA-G1/5 reduces severity of experimental autoimmune uveitis.

Non-infectious uveitis (NIU) is an intractable, recurrent, and painful disease that is a common cause of vision loss. Available treatments of NIU, such as the use of topical corticosteroids, are non-specific and have serious side effects which limits them to short-term use; however, NIU requires long-term treatment to prevent vision loss. Therefore, a single dose therapeutic that mediates long-term immunosuppression with minimal side effects is desirable. In order to develop an effective long-term therapy for NIU, an adeno-associated virus (AAV) gene therapy approach was used to exploit a natural immune tolerance mechanism induced by the human leukocyte antigen G (HLA-G). To mimic the prevention of NIU, naïve Lewis rats received a single intravitreal injection of AAV particles harboring codon-optimized cDNAs encoding HLA-G1 and HLA-G5 isoforms one week prior to the induction of experimental autoimmune uveitis (EAU). AAV-mediated expression of the HLA-G-1 and -5 transgenes in the targeted ocular tissues following a single intravitreal injection of AAV-HLA-G1/5 significantly decreased clinical and histopathological inflammation scores compared to untreated EAU eyes (p < 0.04). Thus, localized ocular gene delivery of AAV-HLA-G1/5 may reduce the off-target risks and establish a long-term immunosuppressive effect that would serve as an effective and novel therapeutic strategy for NIU, with the potential for applications to additional ocular immune-mediated diseases.

Gene Delivery to Human Limbal Stem Cells Using Viral Vectors.

Limbal stem cell (LSC) transplantation is a promising treatment for ocular surface diseases especially LSC deficiency. Genetic engineering represents an attractive strategy to increase the potential for success in LSC transplantations either by correcting autologous diseased LSCs or by decreasing the immunogenicity of allogeneic LSCs. Therefore, two popular viral vectors, adeno-associated viral (AAV) vector and lentiviral (LV) vector, were compared for gene delivery in human LSCs. Transduction efficiency was evaluated by flow cytometry, quantitation of viral genomes, and fluorescence microscopy after introducing eight self-complementary AAV serotypes or LV carrying a green fluorescent protein (GFP) cassette to fresh limbal epithelial cells, cultivated LSC colonies, or after corneal intrastromal injection into human explant tissue. For fresh limbal epithelial cells, AAV6 showed the highest transduction efficiency, followed by LV and AAV4 at 24 h after vector incubation, which did not directly correlate with internalized genome copy number. The colony formation efficiency, as well as colony size over time, showed no significant differences among AAV serotypes, LV, and nontreated controls. The percentage of GFP+ colonies at 14 days post-seeding was significantly higher in the LV group, which plateaued at 50% GFP+ upon serial passages. Interestingly, AAV6-treated colonies initially showed a variegated transduction phenotype with no GFP+ colonies in serial passages. Quantitative polymerase chain reaction and AAV6 capsid staining revealed that transduction was restricted to differentiated cells of LSC colonies at a post-entry step. Following central intrastromal injection of human corneas, both LV and AAV6 transduced the stroma and endothelial cells, and AAV6 also transduced cells of the epithelia. However, no transduction was observed in derived LSC colonies. The collective results demonstrate the effectiveness of LV for stable human LSC genetic engineering and an unreported phenomenon of AAV6 transduction restriction in multipotent cells derived from the human limbus.

Serotype survey of AAV gene delivery via subconjunctival injection in mice.

AAV gene therapy approaches in the posterior eye resulted in the first FDA-approved gene therapy-based drug. However, application of AAV vectorology to the anterior eye has yet to enter even a Phase I trial. Furthermore, the simple and safe subconjunctival injection has been relatively unexplored in regard to AAV vector transduction. To determine the utility of this route for the treatment of various ocular disorders, a survey of gene delivery via natural AAV serotypes was performed and correlated to reported cellular attachment factors. AAV serotypes packaged with a self-complementary reporter were administered via subconjunctival injection to WT mice. Subconjunctival injection of AAV vectors was without incidence; however, vector shedding in tears was noted weeks following administration. AAV transduction was serotype dependent in anterior segment tissues including the eye lid, conjunctiva, and cornea, as well as the periocular tissues including muscle. Transgene product in the cornea was highest for AAV6 and AAV8, however, their corneal restriction was remarkably different; AAV6 appeared restricted to the endothelium layer while AAV8 efficiently transduced the stromal layer. Reported AAV cellular receptors were not well correlated to vector transduction; although, in some cases they were conserved among mouse and human ocular tissues. Subconjunctival administration of particular AAV serotypes may be a simple and safe targeted gene delivery route for ocular surface, muscular, corneal, and optic nerve diseases.

Evaluation of the PEG Density in the PEGylated Chitosan Nanoparticles as a Drug Carrier for Curcumin and Mitoxantrone.

Polyethylene glycolated (PEGylated)curcumin-grafted-chitosan (PCC) conjugates were synthesized with three PEG/chitosan feed molar ratios (1/5, 1/7.5, and 1/10), namely PCC1, PCC2 and PCC3. Chemical structures of these conjugates were characterized by Fourier transform infrared (FTIR) and proton nuclear magnetic resonance (¹H NMR). The degrees of substitution (DS) of PEG were 0.75%, 0.45% and 0.33%, respectively, for PCC1, PCC2 and PCC3by ¹H NMR analysis. Self-assembled PCC nanoparticles (NPs) were spherical as observed in transmission electron microscope images. Mitoxantrone (MTO)-loaded PCC NPs were prepared to analyze the particle size, zeta potential, drug loading, drug release and in vitro cytotoxicity. The MTO-loaded PCC3 NP (DS = 0.33%) possessed the smallest size (~183.1 nm), highest zeta potential (~+34.0 mV) and the largest loading capacity of curcumin (CUR, ~16.1%) and MTO (~8.30%). The release results showed that MTO-loaded PCC3 NP demonstrated the lowest percentage of MTO release and increased as pH decreased, but the CUR release could only be detected at pH 4.0. In the cytotoxicity study, MTO-loaded PCC3 NP displayed the highest cytotoxicity in HepG2 cell line and the best synergistic effect among the tested NPs. Our results suggest that the DS of PEG has impacts on the structures and functions of PCC NPs: the smaller DS of PEG was associated with the smaller size, the higher zeta potential, the slower drug release, and the higher cytotoxicity of NPs.

Cerasomal Lovastatin Nanohybrids for Efficient Inhibition of Triple-Negative Breast Cancer Stem Cells To Improve Therapeutic Efficacy.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a higher risk in younger women and a poorer prognosis and without targeted therapies available currently. Cancer stem cells (CSCs) are increasingly recognized as the main cause of treatment failure and tumor recurrence. The present paper reports the encapsulation of lovastatin (LV) into cerasomes. Compared with free LV, cerasome-encapsulated LV (C-LV) nanohybrids showed cytotoxicity to MDA-MB-231 CSCs in a dose- and time-dependent manner. Furthermore, intravenous injection of C-LV nanohybrids resulted in a significant tumor size reduction in a dose-dependent manner in xenograft tumors derived from subcutaneous inoculation of MDA-MB-231 cells. Furthermore, histopathological and/or immunohistochemical analysis revealed that C-LV nanohybrids significantly induced mammary gland formation and apoptosis and inhibited angiogenesis, the CSC phenotype, and the epithelial-to-mesenchymal transition in xenograft tumors. Most importantly, C-LV nanohybrids were found to be more effective than free LV in inhibiting the growth of breast cancer xenografts and the stemness properties in vivo. To the best of our knowledge, ours is the first demonstration of nanohybrids for efficient inhibition of CSCs derived from TNBC, offering a new option for the TNBC treatment.

Adeno-Associated Virus Vectors and Stem Cells: Friends or Foes?

The infusion of healthy stem cells into a patient-termed "stem-cell therapy"-has shown great promise for the treatment of genetic and non-genetic diseases, including mucopolysaccharidosis type 1, Parkinson's disease, multiple sclerosis, numerous immunodeficiency disorders, and aplastic anemia. Stem cells for cell therapy can be collected from the patient (autologous) or collected from another "healthy" individual (allogeneic). The use of allogenic stem cells is accompanied with the potentially fatal risk that the transplanted donor T cells will reject the patient's cells-a process termed "graft-versus-host disease." Therefore, the use of autologous stem cells is preferred, at least from the immunological perspective. However, an obvious drawback is that inherently as "self," they contain the disease mutation. As such, autologous cells for use in cell therapies often require genetic "correction" (i.e., gene addition or editing) prior to cell infusion and therefore the requirement for some form of nucleic acid delivery, which sets the stage for the AAV controversy discussed herein. Despite being the most clinically applied gene delivery context to date, unlike other more concerning integrating and non-integrating vectors such as retroviruses and adenovirus, those based on adeno-associated virus (AAV) have not been employed in the clinic. Furthermore, published data regarding AAV vector transduction of stem cells are inconsistent in regards to vector transduction efficiency, while the pendulum swings far in the other direction with demonstrations of AAV vector-induced toxicity in undifferentiated cells. The variation present in the literature examining the transduction efficiency of AAV vectors in stem cells may be due to numerous factors, including inconsistencies in stem-cell collection, cell culture, vector preparation, and/or transduction conditions. This review summarizes the controversy surrounding AAV vector transduction of stem cells, hopefully setting the stage for future elucidation and eventual therapeutic applications.