Natural Adeno-Associated Virus Serotypes and Engineered Adeno-Associated Virus Capsid Variants: Tropism Differences and Mechanistic Insights.

Today, adeno-associated virus (AAV)-based vectors are arguably the most promising in vivo gene delivery vehicles for durable therapeutic gene expression. Advances in molecular engineering, high-throughput screening platforms, and computational techniques have resulted in a toolbox of capsid variants with enhanced performance over parental serotypes. Despite their considerable promise and emerging clinical success, there are still obstacles hindering their broader use, including limited transduction capabilities, tissue/cell type-specific tropism and penetration into tissues through anatomical barriers, off-target tissue biodistribution, intracellular degradation, immune recognition, and a lack of translatability from preclinical models to clinical settings. Here, we first describe the transduction mechanisms of natural AAV serotypes and explore the current understanding of the systemic and cellular hurdles to efficient transduction. We then outline progress in developing designer AAV capsid variants, highlighting the seminal discoveries of variants which can transduce the central nervous system upon systemic administration, and, to a lesser extent, discuss the targeting of the peripheral nervous system, eye, ear, lung, liver, heart, and skeletal muscle, emphasizing their tissue and cell specificity and translational promise. In particular, we dive deeper into the molecular mechanisms behind their enhanced properties, with a focus on their engagement with host cell receptors previously inaccessible to natural AAV serotypes. Finally, we summarize the main findings of our review and discuss future directions.

Clinical Application of Liquid Biopsy in Pancreatic Cancer: A Narrative Review.

Pancreatic ductal adenocarcinoma contributes significantly to global cancer-related deaths, featuring only a 10% survival rate over five years. The quest for novel tumor markers is critical to facilitate early diagnosis and tailor treatment strategies for this disease, which is key to improving patient outcomes. In pancreatic ductal adenocarcinoma, these markers have been demonstrated to play a crucial role in early identification, continuous monitoring, and prediction of its prognosis and have led to better patient outcomes. Nowadays, biopsy specimens serve to ascertain diagnosis and determine tumor type. However, liquid biopsies present distinct advantages over conventional biopsy techniques. They offer a noninvasive, easily administered procedure, delivering insights into the tumor's status and facilitating real-time monitoring. Liquid biopsies encompass a variety of elements, such as circulating tumor cells, circulating tumor DNA, extracellular vesicles, microRNAs, circulating RNA, tumor platelets, and tumor endothelial cells. This review aims to provide an overview of the clinical applications of liquid biopsy as a technique in the management of pancreatic cancer.

Extracellular Vesicle-Encapsulated Adeno-Associated Viruses for Therapeutic Gene Delivery to the Heart.

BACKGROUND: Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a significant challenge to its successful clinical use is preexisting neutralizing antibodies (NAbs), which bind to free AAVs, prevent efficient gene transduction, and reduce or negate therapeutic effects. Here we describe extracellular vesicle-encapsulated AAVs (EV-AAVs), secreted naturally by AAV-producing cells, as a superior cardiac gene delivery vector that delivers more genes and offers higher NAb resistance. METHODS: We developed a 2-step density-gradient ultracentrifugation method to isolate highly purified EV-AAVs. We compared the gene delivery and therapeutic efficacy of EV-AAVs with an equal titer of free AAVs in the presence of NAbs, both in vitro and in vivo. In addition, we investigated the mechanism of EV-AAV uptake in human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and mouse models in vivo using a combination of biochemical techniques, flow cytometry, and immunofluorescence imaging. RESULTS: Using cardiotropic AAV serotypes 6 and 9 and several reporter constructs, we demonstrated that EV-AAVs deliver significantly higher quantities of genes than AAVs in the presence of NAbs, both to human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and to mouse hearts in vivo. Intramyocardial delivery of EV-AAV9-sarcoplasmic reticulum calcium ATPase 2a to infarcted hearts in preimmunized mice significantly improved ejection fraction and fractional shortening compared with AAV9-sarcoplasmic reticulum calcium ATPase 2a delivery. These data validated NAb evasion by and therapeutic efficacy of EV-AAV9 vectors. Trafficking studies using human induced pluripotent stem cell-derived cells in vitro and mouse hearts in vivo showed significantly higher expression of EV-AAV6/9-delivered genes in cardiomyocytes compared with noncardiomyocytes, even with comparable cellular uptake. Using cellular subfraction analyses and pH-sensitive dyes, we discovered that EV-AAVs were internalized into acidic endosomal compartments of cardiomyocytes for releasing and acidifying AAVs for their nuclear uptake. CONCLUSIONS: Together, using 5 different in vitro and in vivo model systems, we demonstrate significantly higher potency and therapeutic efficacy of EV-AAV vectors compared with free AAVs in the presence of NAbs. These results establish the potential of EV-AAV vectors as a gene delivery tool to treat heart failure.

Hydroxylation of N-acetylneuraminic Acid Influences the in vivo Tropism of N-linked Sialic Acid-Binding Adeno-Associated Viruses AAV1, AAV5, and AAV6.

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy. However, a number of recent preclinical large animal studies failed to translate into the clinic. This illustrates the formidable challenge of choosing the animal models that promise the best chance of a successful translation into the clinic. Several of the most common AAV serotypes use sialic acid (SIA) as their primary receptor. However, in contrast to most mammals, humans lack the enzyme CMAH, which hydroxylates cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac) into cytidine monophosphate-N-glycolylneuraminic acid (CMP-Neu5Gc). As a result, human glycans only contain Neu5Ac and not Neu5Gc. Here, we investigate the tropism of AAV1, 5, 6 and 9 in wild-type C57BL/6J (WT) and CMAH knock-out (CMAH-/-) mice. All N-linked SIA-binding serotypes (AAV1, 5 and 6) showed significantly lower transduction of the heart in CMAH-/- when compared to WT mice (5-5.8-fold) and, strikingly, skeletal muscle transduction by AAV5 was almost 30-fold higher in CMAH-/- compared to WT mice. Importantly, the AAV tropism or distribution of expression among different organs was also affected. For AAV1, AAV5 and AAV6, expression in the heart compared to the liver was 4.6-8-fold higher in WT than in CMAH-/- mice, and for AAV5 the expression in the heart compared to the skeletal muscle was 57.3-fold higher in WT than in CMAH-/- mice. These data thus strongly suggest that the relative abundance of Neu5Ac and Neu5Gc plays a role in AAV tropism, and that results obtained in commonly used animal models might not translate into the clinic.

Human Adenovirus Serotype 5 Is Sensitive to IgM-Independent Neutralization In Vitro and In Vivo.

Human adenovirus 5 (HAdV-5) is used as a vector in gene therapy clinical trials, hence its interactions with the host immune system have been widely studied. Previous studies have demonstrated that HAdV-5 binds specifically to murine coagulation factor X (mFX), inhibiting IgM and complement-mediated neutralization. Here, we examined the physical binding of immune components to HAdV-5 by nanoparticle tracking analysis, neutralization assays, mass spectrometry analysis and in vivo experiments. We observed that purified mouse Immunoglobulin M (IgM) antibodies bound to HAdV-5 only in the presence of complement components. Active serum components were demonstrated to bind to HAdV-5 in the presence or absence of mFX, indicating that immune molecules and mFX might bind to different sites. Since binding of mFX to HAdV-5 blocks the neutralization cascade, these findings suggested that not all complement-binding sites may be involved in virion neutralization. Furthermore, the data obtained from serum neutralization experiments suggested that immune molecules other than IgM and IgG may trigger activation of the complement cascade in vitro. In vivo experiments were conducted in immunocompetent C57BL/6 or immuno-deficient Rag2-/- mice. HAdV-5T* (a mutant HAdV-5 unable to bind to human or mFX) was neutralized to some extent in both mouse models, suggesting that murine immunoglobulins were not required for neutralization of HAdV-5 in vivo. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis of HAdV-5 and HAdV-5T* after exposure to murine sera showed stable binding of C3 and C4b in the absence of mFX. In summary, these results suggest that HAdV-5 neutralization can be mediated by both the classical and alternative pathways and that, in the absence of immunoglobulins, the complement cascade can be activated by direct binding of C3 to the virion.

AAV Vectors for Efficient Gene Delivery to Rodent Hearts.

Currently, gene therapy is one of the most promising fields in biomedicine, with great therapeutic potential for an array of inherited and acquired diseases. Adeno-associated viral (AAV) vectors have emerged as promising tools to deliver selectively a therapeutic payload to target organs, including the heart. In this chapter, we describe the production and quality control of recombinant AAV (rAAV) vectors of the serotype 9, the most cardiotropic AAV serotype when delivered systemically in rodents. We also describe the systemic administration of rAAV vectors and the local delivery of rAAV vectors by direct intramyocardial injection. Taken together, the methods described in this chapter will allow the reader to deliver efficiently therapeutic genes to the rodent heart, both globally and regionally.

Defining a Novel Role for the Coxsackievirus and Adenovirus Receptor in Human Adenovirus Serotype 5 Transduction In Vitro in the Presence of Mouse Serum.

Human adenoviral serotype 5 (HAdV-5) vectors have predominantly hepatic tropism when delivered intravascularly, resulting in immune activation and toxicity. Coagulation factor X (FX) binding to HAdV-5 mediates liver transduction and provides protection from virion neutralization in mice. FX is dispensable for liver transduction in mice lacking IgM antibodies or complement, suggesting that alternative transduction pathways exist. To identify novel factor(s) mediating HAdV-5 FX-independent entry, we investigated HAdV-5 transduction in vitro in the presence of serum from immunocompetent C57BL/6 or immunocompromised mice lacking IgM antibodies (Rag 2-/- and NOD-scid-gamma [NSG]). Sera from all three mouse strains enhanced HAdV-5 transduction of A549 cells. While inhibition of HAdV-5-FX interaction with FX-binding protein (X-bp) inhibited transduction in the presence of C57BL/6 serum, it had negligible effect on the enhanced transduction observed in the presence of Rag 2-/- or NSG serum. Rag 2-/- serum also enhanced transduction of the FX binding-deficient HAdV-5HVR5*HVR7*E451Q (AdT*). Interestingly, Rag 2-/- serum enhanced HAdV-5 transduction in a FX-independent manner in CHO-CAR and SKOV3-CAR cells (CHO or SKOV3 cells transfected to stably express human coxsackievirus and adenovirus receptor [CAR]). Additionally, blockade of CAR with soluble HAdV-5 fiber knob inhibited mouse serum-enhanced transduction in A549 cells, suggesting a potential role for CAR. Transduction of HAdV-5 KO1 and HAdV-5/F35 (CAR binding deficient) in the presence of Rag 2-/- serum was equivalent to that of HAdV-5, indicating that direct interaction between HAdV-5 and CAR is not required. These data suggest that FX may protect HAdV-5 from neutralization but has minimal contribution to HAdV-5 transduction in the presence of immunocompromised mouse serum. Alternatively, transduction occurs via an unidentified mouse serum protein capable of bridging HAdV-5 to CAR.IMPORTANCE The intravascular administration of HAdV-5 vectors can result in acute liver toxicity, transaminitis, thrombocytopenia, and injury to the vascular endothelium, illustrating challenges yet to overcome for HAdV-5-mediated systemic gene therapy. The finding that CAR and potentially an unidentified factor present in mouse serum might be important mediators of HAdV-5 transduction highlights that a better understanding of the complex biology defining the interplay between adenovirus immune recognition and cellular uptake mechanisms is still required. These findings are important to inform future optimization and development of HAdV-5-based adenoviral vectors for gene therapy.

"Effectiveness of continuous vertebral resonant oscillation using the POLD method in the treatment of lumbar disc hernia". A randomized controlled pilot study.

This study analyses the efficacy of manual oscillatory therapy, following the POLD technique, for acute Lumbar Disc Hernia (LDH) and compares it to usual treatment. A randomised, controlled, triple-blind pilot clinical trial. The sample of 30 patients was divided into two homogeneous groups to receive usual treatment (A) or treatment with the POLD technique (B). We analysed range of motion and subjective variables such as the severity (visual analogue pain scale (VAS)) and extension of the pain. With the application of POLD therapy, patients presented significant changes on range of motion (forward flexion with p < 0.05) at completion of the trial in comparison with the control group. They showed a significant reduction in the severity of pain with a mean VAS scale for lumbar, glutaeus and thigh pain, which improved from 5.09 to 0.79, 5.07 to 0.97 and 4.43 to 0.49 respectively (p < 0.05), and also when compared to usual treatment (p < 0.05) for all body regions. Moreover, we observed a reduction in pain extension (centralization phenomena) (p < 0.001) in comparison with usual treatment. In our study the POLD Method was shown to be an effective manual therapy approach for reducing the severity and irradiation of the pain in LDH patients with sciatica, and more efficient than usual treatment.

The importance of coagulation factors binding to adenovirus: historical perspectives and implications for gene delivery.

INTRODUCTION: The interaction of human adenovirus (HAdV) serotype 5 (HAdV-5) with the blood coagulation factor X (FX) results in a high liver transduction after AdV intravascular administration, causing toxicity and limiting AdV delivery to the target tissue. However, FX also protects adenoviral vectors from neutralization by the complement system and natural antibodies, potentially benefiting adenoviral gene delivery, as neutralization results in the reduction of HAdV-5 binding to host cells. AREAS COVERED: This review gives an overview on the use of adenoviruses as gene transfer vehicles and their impact in gene therapy. The structure of coagulation factors and their interactions with HAdV are described, highlighting their influence on the AdV biodistribution profile. The implications of this interaction in immunity and its potential influence on the use of adenoviral vectors in gene therapy applications are discussed. EXPERT OPINION: The protective role of FX brings under discussion how beneficial abolishment of FX binding would be for HAdV-5 liver detargeting. The dispensability of FX for liver transduction in immunocompromised mice suggests the involvement of other blood factors or receptors in enhancing HAdV-5 liver transduction. Better understanding of the interactions that take place in the bloodstream is essential to generate safe and efficient adenoviral vectors.

Circumventing antivector immunity: potential use of nonhuman adenoviral vectors.

Adenoviruses are efficient gene delivery vectors based on their ability to transduce a wide variety of cell types and drive high-level transient transgene expression. While there have been advances in modifying human adenoviral (HAdV) vectors to increase their safety profile, there are still pitfalls that need to be further addressed. Preexisting humoral and cellular immunity against common HAdV serotypes limits the efficacy of gene transfer and duration of transgene expression. As an alternative, nonhuman AdV (NHAdV) vectors can circumvent neutralizing antibodies against HAdVs in immunized mice and monkeys and in human sera, suggesting that NHAdV vectors could circumvent preexisting humoral immunity against HAdVs in a clinical setting. Consequently, there has been an increased interest in developing NHAdV vectors for gene delivery in humans. In this review, we outline the recent advances and limitations of HAdV vectors for gene therapy and describe examples of NHAdV vectors focusing on their immunogenicity, tropism, and potential as effective gene therapy vehicles.