ABSTRACT
Introduction: The procurement of medicines via China's national volume-based procurement (NVBP) necessitates collaboration among various entities. This paper highlights the legal significance of the engagement of pharmaceutical companies, hospitals, and the National Healthcare Security Administration (NHSA) in improving drug accessibility. Methods: We conducted a numerical simulation using MATLAB to develop an evolutionary game model involving these three participants in NVBP. Results: Our findings indicate that the final evolutionary stabilization strategies are pharmaceutical companies actively participating, hospitals using bid-winning medicines, and the NHSA implementing a low-intensity intervention. The study reveals that the evolutionary outcomes for hospitals and pharmaceutical companies are significantly affected by factors such as NHSA's subsidy level and pharmaceutical companies' level of participation. However, NHSA's decision-making process is less influenced by these factors. Discussion: From a legal perspective, the successful implementation of NVBP, ensuring fairness and legality, requires adherence to relevant policies and regulations. The NHSA should employ statutory incentives and regulatory methods in formulating and adjusting NVBP policy to enable pharmaceutical companies, hospitals, and the NHSA to exercise their rights rationally within the legal framework of the game process.
Subject(s)
Drug Industry , China , Humans , Health Services Accessibility , HospitalsABSTRACT
Human bocavirus 1 (HBoV1) is a human parvovirus that causes lower respiratory tract infections in young children. It contains a single-stranded (ss) DNA genome of ~5.5 kb that encodes a small noncoding RNA of 140 nucleotides known as bocavirus-encoded small RNA (BocaSR), in addition to viral proteins. Here, we determined the secondary structure of BocaSR in vivo by using DMS-MaPseq. Our findings reveal that BocaSR undergoes N6-methyladenosine (m6A) modification at multiple sites, which is critical for viral DNA replication in both dividing HEK293 cells and nondividing cells of the human airway epithelium. Mechanistically, we found that m6A-modified BocaSR serves as a mediator for recruiting Y-family DNA repair DNA polymerase (Pol) η and Pol κ likely through a direct interaction between BocaSR and the viral DNA replication origin at the right terminus of the viral genome. Thus, this report represents direct involvement of a viral small noncoding RNA in viral DNA replication through m6A modification.
Subject(s)
Adenosine , DNA Replication , DNA, Viral , DNA-Directed DNA Polymerase , RNA, Viral , Virus Replication , Humans , Adenosine/analogs & derivatives , Adenosine/metabolism , Virus Replication/genetics , DNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/genetics , DNA, Viral/genetics , DNA, Viral/metabolism , HEK293 Cells , RNA, Viral/genetics , RNA, Viral/metabolism , Human bocavirus/genetics , Human bocavirus/metabolism , Genome, Viral/genetics , Parvoviridae Infections/virologyABSTRACT
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with F508del being the most prevalent mutation. The combination of CFTR modulators (potentiator and correctors) has provided benefit to CF patients carrying the F508del mutation; however, the safety and effectiveness of in utero combination modulator therapy remains unclear. We created a F508del ferret model to test whether ivacaftor/lumacaftor (VX-770/VX-809) therapy can rescue in utero and postnatal pathologies associated with CF. Using primary intestinal organoids and air-liquid interface cultures of airway epithelia, we demonstrate that the F508del mutation in ferret CFTR results in a severe folding and trafficking defect, which can be partially restored by treatment with CFTR modulators. In utero treatment of pregnant jills with ivacaftor/lumacaftor prevented meconium ileus at birth in F508del kits and sustained postnatal treatment of CF offspring improved survival and partially protected from pancreatic insufficiency. Withdrawal of ivacaftor/lumacaftor treatment from juvenile CF ferrets reestablished pancreatic and lung diseases, with altered pulmonary mechanics. These findings suggest that in utero intervention with a combination of CFTR modulators may provide therapeutic benefits to individuals with F508del. This CFTR-F508del ferret model may be useful for testing therapies using clinically translatable endpoints.
Subject(s)
Aminophenols , Aminopyridines , Benzodioxoles , Cystic Fibrosis Transmembrane Conductance Regulator , Cystic Fibrosis , Ferrets , Quinolones , Animals , Female , Pregnancy , Aminophenols/therapeutic use , Aminophenols/pharmacology , Aminopyridines/pharmacology , Aminopyridines/therapeutic use , Benzodioxoles/therapeutic use , Benzodioxoles/pharmacology , Chloride Channel Agonists/therapeutic use , Chloride Channel Agonists/pharmacology , Cystic Fibrosis/genetics , Cystic Fibrosis/drug therapy , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Disease Models, Animal , Drug Combinations , Mutation , Quinolones/pharmacology , Quinolones/therapeutic useABSTRACT
The dosing interval for effective recombinant adeno-associated virus (rAAV)-mediated gene therapy of cystic fibrosis lung disease remains unknown. Here, we assessed the durability of rAAV2.5T-fCFTRΔR-mediated transgene expression and neutralizing antibody (NAb) responses in lungs of adult wild-type ferrets. Within the first 3 months following rAAV2.5T-fCFTRΔR delivery to the lung, CFTRΔR transgene expression declined â¼5.6-fold and then remained stable to 5 months at â¼26% the level of endogenous CFTR. rAAV NAbs in the plasma and bronchoalveolar lavage fluid (BALF) peaked at 21 days, coinciding with peak ELISpot T cell responses to AAV capsid peptides, after which both responses declined and remained stable at 4-5 months post dosing. Administration of reporter vector rAAV2.5T-gLuc (gaussia luciferase) at 5 months following rAAV2.5T-fCFTRΔR dosing gave rise to similar levels of gLuc expression in the BALF as observed in age-matched reporter-only controls, demonstrating that residual BALF NAbs were functionally insignificant. Notably, the second vector administration led to a 2.6-fold greater ELISpot T cell response and â¼2.3-fold decline in fCFTRΔR mRNA and vector genomes derived from the initial rAAV2.5T-fCFTRΔR administration, suggesting selective destruction of transduced cells from the first vector dose. These findings provide insights into humoral and cellular immune response to rAAV that may be useful for optimizing gene therapy to the cystic fibrosis lung.
ABSTRACT
Background: Blockchain is expected to mitigate consumers' risk-aversion and quality uncertainty about generic drugs in medicine supply chains. This study investigates the effect of blockchain adoption for disclosing the quality information of generic drugs that compete with original drugs in the market and proposes legal measures accordingly. Methods: We employ a game-theoretic model to analyze a medicine supply chain including a generic drug manufacturer, an original drug manufacturer, and a retailer. We examine when should the supply chain members adopt blockchain for generic drugs and how blockchain affects the medicine supply chain. Results: Our results show that the quality information of generic drugs determines how blockchain adoption affects the price and sales quantity of generic and original drugs. Moreover, we observe that the generic drugs manufacturer and the retailer decide to adopt blockchain only if consumers' risk-aversion degree is sufficiently low. Also, a low risk-aversion degree can lead to higher whole supply chain's profitability with blockchain adoption, and generate a win-win-win situation of blockchain adoption for the consumers, the generic drug manufacturer, and the retailer. Conclusion: To mitigate consumers' risk aversion, the law should safeguard consumer rights. Blockchain adoption can benefit the medicine supply chain and consumers under certain conditions. However, it also requires the coordination of supply chain members' benefits and the disclosure of quality information.
ABSTRACT
IMPORTANCE: The essential steps of successful gene delivery by recombinant adeno-associated viruses (rAAVs) include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63, whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.
Subject(s)
Bronchi , Dependovirus , Epithelium , Gene Transfer Techniques , Genetic Vectors , Transduction, Genetic , Humans , Capsid Proteins/genetics , Capsid Proteins/metabolism , Dependovirus/genetics , Dependovirus/metabolism , DNA , Epithelium/metabolism , Epithelium/virology , Gene Transfer Techniques/trends , Genetic Therapy/methods , Genetic Vectors/genetics , Bronchi/metabolism , Bronchi/virology , Active Transport, Cell Nucleus , Gene Editing/trendsABSTRACT
Recombinant (r)AAV2.5T was selected from the directed evolution of an AAV capsid library in human airway epithelium (HAE). The capsid gene of rAAV2.5T is a chimera of the N-terminal unique coding sequence of AAV2 VP1 unique (VP1u) and the VP2- and VP3-coding sequence of AAV5 with a single amino acid mutation of A581T. We conducted two rounds of genome wide CRISPR gRNA library screening for host factors limiting rAAV2.5T transduction in HeLa S3 cells. The screen identified several genes that are critical for rAAV2.5T transduction in HeLa S3 cells, including previously reported genes KIAA0319L , TM9SF2 , VPS51 , and VPS54 , as well as a novel gene WDR63 . We verified the role of KIAA0319L and WDR63 in rAAV2.5T transduction of polarized HAE by utilizing CRISPR gene knockouts. Although KIAA0319L, a proteinaceous receptor for multiple AAV serotypes, played an essential role in rAAV2.5T transduction of polarized HAE either from apical or basolateral side, our findings demonstrated that the internalization of rAAV2.5T was independent of KIAA0319L. Importantly, we confirmed WDR63 is an important player in rAAV2.5T transduction of HAE, while not being involved in vector internalization and nuclear entry. Furthermore, we identified that the basal stem cells of HAE can be significantly transduced by rAAV2.5T. Significance: The essential steps of a successful gene delivery by rAAV include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63 , whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.
ABSTRACT
Adeno-associated virus 2.5T (AAV2.5T) was selected from the directed evolution of AAV capsid library in human airway epithelia. This study found that recombinant AAV2.5T (rAAV2.5T) transduction of well-differentiated primary human airway epithelia induced a DNA damage response (DDR) characterized by the phosphorylation of replication protein A32 (RPA32), histone variant H2AX (H2A histone family member X), and all three phosphatidylinositol 3-kinase-related kinases: ataxia telangiectasia mutated kinase, ataxia telangiectasia and Rad3-related kinase (ATR), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). While suppressing the expression of ATR by a specific pharmacological inhibitor or targeted gene silencing inhibited rAAV2.5T transduction, DNA-PKcs inhibition or targeted gene silencing significantly increased rAAV2.5T transgene expression. Notably, DNA-PKcs inhibitors worked as a "booster" to further increase rAAV2.5T transgene expression after treatment with doxorubicin and did not compromise epithelial integrity. Thus, our study provides evidence that DDR is associated with rAAV transduction in well-differentiated human airway epithelia, and DNA-PKcs inhibition has the potential to boost rAAV transduction. These findings highlight that the application of DDR inhibition-associated pharmacological interventions has the potential to increase rAAV transduction and thus to reduce the required vector dose.
ABSTRACT
The efficacy of redosing the recombinant adeno-associated virus (rAAV) vector rAAV2.5T to ferret lung is limited by AAV neutralizing antibody (NAb) responses. While immunosuppression strategies have allowed for systemic rAAV repeat dosing, their utility for rAAV lung-directed gene therapy is largely unexplored. To this end, we evaluated two immunosuppression (IS) strategies to improve repeat dosing of rAAV2.5T to ferret lungs: (1) a combination of three IS drugs (Tri-IS) with broad coverage against cellular and humoral responses (methylprednisolone [MP], azathioprine, and cyclosporine) and (2) MP alone, which is typically used in systemic rAAV applications. Repeat dosing utilized AAV2.5T-SP183-fCFTRΔR (recombinant ferret CFTR transgene), followed 28 days later by AAV2.5T-SP183-gLuc (for quantification of transgene expression). Both the Tri-IS and MP strategies significantly improved transgene expression following repeat dosing and reduced AAV2.5T NAb responses in the bronchioalveolar lavage fluid (BALF) and plasma, while AAV2.5T binding antibody subtypes and cellular immune responses by ELISpot were largely unchanged by IS. One exception was the reduction in plasma AAV2.5T binding immunoglobulin G (IgG) in both IS groups. Only the Tri-IS strategy significantly suppressed splenocyte expression of IFNA (interferon α [IFN-α]) and IL4. Our studies suggest that IS strategies may be useful in clinical application of rAAV targeting lung genetic diseases such as cystic fibrosis.
ABSTRACT
Adeno-associated virus (AAV) belongs to the Dependoparvovirus genus of the Parvoviridae family. AAV replication relies on a helper virus, such as adenovirus (Ad). Co-infection of AAV and Ad induces a DNA damage response (DDR), although its function in AAV DNA replication remains unknown. In this study, monoinfection of AAV2 in HEK293T cells expressing a minimal set of Ad helper genes was used to investigate the role of the DDR solely induced by AAV. We found that AAV2 DNA replication, but not single stranded (ss)DNA genome accumulation and Rep expression only, induced a robust DDR in HEK293T cells. The induced DDR featured the phosphorylation of replication protein A32 (RPA32), histone variant H2AX (H2A histone family member X), and all 3 phosphatidylinositol 3-kinase-related kinases (PIKKs). We also found that the kinase ataxia telangiectasia and Rad3-related protein (ATR) plays a major role in AAV2 DNA replication and that Y family DNA repair DNA polymerases η (Pol η) and Pol κ contribute to AAV2 DNA replication both in vitro and in HEK293T cells. Knockout of Pol η and Pol κ in HEK293T cells significantly decreased wild-type AAV2 replication and recombinant AAV2 production. Thus, our study has proven that AAV2 DNA replication induces a DDR, which in turn initiates a DNA repairing process that partially contributes to the viral genome amplification in HEK293T cells. IMPORTANCE Recombinant AAV (rAAV) has emerged as one of the preferred delivery vectors for clinical gene therapy. rAAV production in HEK293 cells by transfection of a rAAV transgene plasmid, an AAV Rep and Cap expression packaging plasmid, and an Ad helper plasmid remains the popular method. Here, we demonstrated that the high fidelity Y family DNA repair DNA polymerase, Pol η, and Pol κ, plays a significant role in AAV DNA replication and rAAV production in HEK293T cells. Understanding the AAV DNA replication mechanism in HEK293T cells could provide clues to increase rAAV vector yield produced from the transfection method. We also provide evidence that the ATR-mediated DNA repair process through Pol η and Pol κ is one of the mechanisms to amplify AAV genome, which could explain AAV replication and rAAV ssDNA genome conversion in mitotic quiescent cells.
Subject(s)
Histones , Virus Replication , Humans , Histones/genetics , Dependovirus/genetics , DNA Replication , HEK293 Cells , DNA, Viral/genetics , DNA Repair , DNA Damage , Genetic VectorsABSTRACT
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a chronic disease that affects multiple organs, including the lung. We developed a CF ferret model of a scarless G551âD substitution in CFTR (CFTRG551D-KI), enabling approaches to correct this gating mutation in CF airways via gene editing. Homology-directed repair (HDR) was tested in Cas9-expressing CF airway basal cells (Cas9-GKI) from this model, as well as reporter basal cells (Y66S-Cas9-GKI) that express an integrated nonfluorescent Y66S-EGFP (enhanced green fluorescent protein) mutant gene to facilitate rapid assessment of HDR by the restoration of fluorescence. Recombinant adeno-associated virus (rAAV) vectors were used to deliver two DNA templates and sgRNAs for dual-gene editing at the EGFP and CFTR genes, followed by fluorescence-activated cell sorting of EGFPY66S-corrected cells. When gene-edited airway basal cells were polarized at an air-liquid interface, unsorted and EGFPY66S-corrected sorted populations gave rise to 26.0% and 70.4% CFTR-mediated Cl- transport of that observed in non-CF cultures, respectively. The consequences of gene editing at the CFTRG551D locus by HDR and nonhomologous end joining (NHEJ) were assessed by targeted gene next-generation sequencing (NGS) against a specific amplicon. NGS revealed HDR corrections of 3.1% of G551 sequences in the unsorted population of rAAV-infected cells, and 18.4% in the EGFPY66S-corrected cells. However, the largest proportion of sequences had indels surrounding the CRISPR (clustered regularly interspaced short palindromic repeats) cut site, demonstrating that NHEJ was the dominant repair pathway. This approach to simultaneously coedit at two genomic loci using rAAV may have utility as a model system for optimizing gene-editing efficiencies in proliferating airway basal cells through the modulation of DNA repair pathways in favor of HDR.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator , Cystic Fibrosis , Animals , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Dependovirus/genetics , Dependovirus/metabolism , Ferrets/genetics , Ferrets/metabolism , Genetic Vectors/genetics , Cystic Fibrosis/genetics , Cystic Fibrosis/therapy , Mutation , Lung/metabolism , DNAABSTRACT
Women with intellectual disabilities (WIDs) are entitled to the rights to marriage and reproduction under Chinese law. However, Chinese marriage and reproduction regimes have imposed restrictions on their right to marry that limit the exercise of their reproductive rights. Marrying WIDs has become a popular choice for men in rural areas. Although in China's Civil Code, the legislature has repealed the prior requirement of a medical examination for marriage registration, the exercise of rights to marriage and reproduction by WIDs is still restricted. China has not fully implemented equal rights for persons with disabilities under the United Nations Convention on the Rights of Persons with Disabilities. China needs to set legal procedural requirements for the exercise of these rights for different types of WIDs, systematically constructing them at three levels: legal basis, legislative implementation, and institutional support.
ABSTRACT
Alpha-1 antitrypsin deficiency (AATD) is the most common genetic cause and risk factor for chronic obstructive pulmonary disease, but the field lacks a large-animal model that allows for longitudinal assessment of pulmonary function. We hypothesized that ferrets would model human AATD-related lung and hepatic disease. AAT-knockout (AAT-KO) and PiZZ (E342K, the most common mutation in humans) ferrets were generated and compared with matched controls using custom-designed flexiVent modules to perform pulmonary function tests, quantitative computed tomography (QCT), bronchoalveolar lavage (BAL) proteomics, and alveolar morphometry. Complete loss of AAT (AAT-KO) led to increased pulmonary compliance and expiratory airflow limitation, consistent with obstructive lung disease. QCT and morphometry confirmed emphysema and airspace enlargement, respectively. Pathway analysis of BAL proteomics data revealed inflammatory lung disease and impaired cellular migration. The PiZ mutation resulted in altered AAT protein folding in the liver, hepatic injury, and reduced plasma concentrations of AAT, and PiZZ ferrets developed obstructive lung disease. In summary, AAT-KO and PiZZ ferrets model the progressive obstructive pulmonary disease seen in AAT-deficient patients and may serve as a platform for preclinical testing of therapeutics including gene therapy.
Subject(s)
Pulmonary Disease, Chronic Obstructive , Pulmonary Emphysema , alpha 1-Antitrypsin Deficiency , Animals , Ferrets , Humans , Lung/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , alpha 1-Antitrypsin Deficiency/complications , alpha 1-Antitrypsin Deficiency/genetics , alpha 1-Antitrypsin Deficiency/therapyABSTRACT
Personal purchases of novel coronavirus antigen detection reagents (ADRs) for self-detection have contributed to the optimization of medical resources and containment of the COVID-19 pandemic. The recurring occurrence of false testing results in China has generated concerns regarding the quality of ADRs and the testing mechanism for medical devices. Academic viewpoints and remarks on the sensitivity, application possibilities, and product innovation of ADRs may be found in the extant scientific literature. However, the current research does not explore the microscopic product quality concerns that emerge throughout the production and marketing of ADRs. To explore strategic equilibrium circumstances and behavioral evolution processes, an evolutionary game model was developed to include ADR manufacturers, third-party medical device inspection agencies, and regulatory authorities. The results reveal that the quantity of illegal incentives, the cost of regulation, and the loss of government credibility have a major impact on the decisions of regulatory authorities and determine three potential systemic equilibrium states. To maximize social welfare, ADRs should be incorporated into China's medication price monitoring system in order to manage market prices. To cut regulatory expenses, the government should employ blockchain technology for traceable network regulation of ADR product quality. The government should also protect the people's right to free speech and encourage online reporting of adverse incidents caused by ADRs. The conclusions of this article can provide many developing nations with important insights for regulating the quality of ADR products.
Subject(s)
COVID-19 , Pandemics , Humans , COVID-19/diagnosis , COVID-19/epidemiology , China/epidemiology , GovernmentABSTRACT
Vaccination against SARS-CoV-2 has been the most significant measure in the preventing and controlling the spread of COVID-19. The COVID-19 vaccination rate in China is relatively high worldwide. However, the vaccination policies behind China's effective containment of COVID-19 are not well known. Some local governments have mandated vaccination for citizens and limited the freedom of movement and other fundamental rights of the unvaccinated. This paper examines the legitimacy of mandatory vaccination policies and their institutional logic within the Chinese legal framework. China prioritizes collective rights such as health over individual rights such as privacy. However, the mandatory vaccination policies lack statutory authorization and will restrict Chinese citizens' autonomy and compromise their privacy.
ABSTRACT
OBJECTIVE: To investigate the beneficial effect of brain-derived neurotrophic factor (BDNF) -overexpressing human umbilical cord mesenchymal stem cell (hUC-MSC)-derived motor neurons in the human Cu, Zn-superoxide dismutase1 (hSOD1)G93A amyotrophic lateral sclerosis (ALS) mice. METHODS: The BDNF gene was transfected into hUC-MSC-derived motor neurons by the lentivirus-mediated method. hSOD1G93A mice were assigned to the ALS, ALS/MN, and ALS/MN-BDNF groups, and intrathecally administrated phosphate-buffered saline (PBS), motor neurons, or motor neurons overexpressing BDNF, respectively. The control group included non-transgenic wild-type littermates administrated PBS. One month after transplantation, the motor function of the mice was assessed by the rotarod test, and the lumbar enlargements were then isolated to detect the expression of hSOD1 and BDNF by western blotting, and the expression of choline acetyltransferase (ChAT), homeobox protein 9 (HB9), major histocompatibility complex I (MHCI) and microtubule-associated protein-2 (MAP-2) by immunofluorescence assay. RESULTS: After transplantation, mice in the ALS/MN-BDNF and ALS/MN groups both exhibited longer latency to fall and longer survival than those in the ALS group (P < 0.01 vs. P < 0.05), and the improvement was more significant in the former than in the latter. However, cell transplantation did not delay disease onset. In the lumbar enlargements of the ALS/MN-BDNF and ALS/MN groups, the expression of hSOD1 was slightly reduced without statistical significance (P > 0.05), but the expression of BDNF, ChAT and HB9, and the co-expression of MHCI and MAP-2 were significantly greater than in the ALS group (P < 0.01), with the differences also being more prominent in the former group than in the latter. CONCLUSIONS: Transplantation of BDNF-overexpressing hUC-MSC-derived motor neurons can improve motor performance and prolong the survival of hSOD1G93A mice. Combining stem cell-derived motor neurons with BDNF might provide a new therapeutic strategy for ALS.
Subject(s)
Amyotrophic Lateral Sclerosis , Brain-Derived Neurotrophic Factor/metabolism , Motor Neurons/metabolism , Motor Neurons/transplantation , Animals , Humans , Male , Mesenchymal Stem Cells/cytology , Mice , Mice, Transgenic , Motor Activity/physiology , Mutation , Superoxide Dismutase-1/genetics , Umbilical Cord/cytologyABSTRACT
Readministration of recombinant adeno-associated virus (rAAV) may be necessary to treat cystic fibrosis (CF) lung disease using gene therapy. However, little is known about rAAV-mediated immune responses in the lung. Here, we demonstrate the suitability of the ferret for testing AAV2.5T-mediated CFTR delivery to the lung and characterization of neutralizing-antibody (NAb) responses. AAV2.5T-SP183-hCFTRΔR efficiently transduced both human and ferret airway epithelial cultures and complemented CFTR Cl- currents in CF airway cultures. Delivery of AAV2.5T-hCFTRΔR to neonatal and juvenile ferret lungs produced hCFTR mRNA at 200%-300% greater levels than endogenous fCFTR. Single-dose (AAV2.5T-SP183-gLuc) or repeat dosing (AAV2.5T-SP183-fCFTRΔR followed by AAV2.5T-SP183-gLuc) of AAV2.5T was performed in neonatal and juvenile ferrets. Repeat dosing significantly reduced transgene expression (11-fold) and increased bronchoalveolar lavage fluid (BALF) NAbs only in juvenile, but not neonatal, ferrets, despite near-equivalent plasma NAb responses in both age groups. Notably, both age groups demonstrated a reduction in BALF anti-capsid binding immunoglobulin (Ig) G, IgM, and IgA antibodies after repeat dosing. Unique to juvenile ferrets was a suppression of plasma anti-capsid-binding IgM after the second vector administration. Thus, age-dependent immune system maturation and isotype switching may affect the development of high-affinity lung NAbs after repeat dosing of AAV2.5T and may provide a path to blunt AAV-neutralizing responses in the lung.
ABSTRACT
Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air-liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/metabolism , Epithelial Cells/metabolism , Gene Expression Regulation , MicroRNAs/genetics , Respiratory Mucosa/metabolism , Stem Cells/metabolism , Cell Differentiation , Cystic Fibrosis/genetics , Cystic Fibrosis/pathology , Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Genetic Vectors , HEK293 Cells , Humans , Lentivirus/geneticsABSTRACT
The domestic ferret (Mustela putorius furo) has proven to be a useful species for modeling human genetic and infectious diseases of the lung and brain. However, biomedical research in ferrets has been hindered by the lack of rapid and cost-effective methods for genome engineering. Here, we utilized CRISPR/Cas9-mediated, homology-independent insertion at the ROSA26 "safe harbor" locus in ferret zygotes and created transgenic animals expressing a dual-fluorescent Cre-reporter system flanked by PhiC31 and Bxb1 integrase attP sites. Out of 151 zygotes injected with circular transgene-containing plasmid and Cas9 protein loaded with the ROSA26 intron-1 sgRNA, there were 23 births of which 5 had targeted integration events (22% efficiency). The encoded tdTomato transgene was highly expressed in all tissues evaluated. Targeted integration was verified by PCR analyses, Southern blot, and germ-line transmission. Function of the ROSA26-CAG-LoxPtdTomatoStopLoxPEGFP (ROSA-TG) Cre-reporter was confirmed in primary cells following Cre expression. The Phi31 and Bxb1 integrase attP sites flanking the transgene will also enable rapid directional insertion of any transgene without a size limitation at the ROSA26 locus. These methods and the model generated will greatly enhance biomedical research involving lineage tracing, the evaluation of stem cell therapy, and transgenesis in ferret models of human disease.
Subject(s)
Animals, Genetically Modified/genetics , CRISPR-Cas Systems/genetics , Gene Knock-In Techniques/methods , Gene Transfer Techniques , Genetic Engineering/methods , Animals , DNA (Cytosine-5-)-Methyltransferases/genetics , Ferrets , Genes, Reporter/genetics , RNA, Guide, Kinetoplastida/genetics , Repressor Proteins/genetics , Viral Proteins/geneticsABSTRACT
The genome of recombinant adeno-associated virus 2 (rAAV2) remains a promising candidate for gene therapy for cystic fibrosis (CF) lung disease, but due to limitations in the packaging capacity and the tropism of this virus with respect to the airways, strategies have evolved for packaging an rAAV2 genome (up to 5.8 kb) into the capsid of human bocavirus 1 (HBoV1) to produce a chimeric rAAV2/HBoV1 vector. Although a replication-incompetent HBoV1 genome has been established as a trans helper for capsid complementation, this system remains suboptimal with respect to virion yield. Here, a streamlined production system is described based on knowledge of the involvement of HBoV1 nonstructural (NS) proteins NS1, NS2, NS3, NS4, and NP1 in the process of virion production. The analyses reveal that NS1 and NS2 negatively impact virion production, NP1 is required to prevent premature termination of transcription of the cap mRNA from the native genome, and silent mutations within the polyadenylation sites of the cap coding sequence can eliminate this requirement for NP1. It is further shown that preventing the expression of all NS proteins significantly increases virion yield. Whereas the expression of capsid proteins VP1, VP2, and VP3 from a codon-optimized cap mRNA was highly efficient, optimal virion assembly, and thus potency, required enhanced VP1 expression, entailing a separate VP1 expression cassette. The final NS protein-free production system uses three-plasmid co-transfection of HEK293 cells, with one trans helper plasmid encoding VP1 and the AAV2 Rep proteins, and another encoding VP2-3 and components from adenovirus. This system yielded >16-fold more virions than the prototypic system, without reducing transduction potency. This increase in virion production is expected to facilitate greatly both research on the biology of rAAV2/HBoV1 and preclinical studies testing the effectiveness of this vector for gene therapy of CF lung disease in large animal models.