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Tissue distribution of GFP expression following intrapancreatic injection of AAV-GFP serotypes. (A) Whole-body fluorescence images 28 days post-injection of 10 11 VP of each vector were compared with saline injected (control) and transgenic (GFP) mice. Treatment with AAV8 resulted in localized gene expression comparable to the pancreas of GFP transgenic mice (red arrows). Please note the fluorescence on the control and AAV1 mice was reflected from the GFP transgenic mouse. (B) Fluorescent images of individual organs from the above mice under UV light. (C) Microscopic analysis of GFP distribution in tissues collected from the above mice. Note efficient transduction of pancreas and liver by AAV8. Magnification: 10?.
Source publication
Efficient delivery of therapeutic proteins into the pancreas represents a major obstacle to gene therapy of pancreatic disorders. The current study compared the efficiency of recombinant lentivirus and adeno-associated virus (AAV) serotypes 1, 2, 5, 8 vectors delivered by intrapancreatic injection for gene transfer in vivo. Our results indicate tha...
Contexts in source publication
Context 1
... number 9159 Figure 2A). Various 296 tissues were then dissected and placed on the UV 297 transluminator. ...
Context 2
... 296 tissues were then dissected and placed on the UV 297 transluminator. In line with the whole-body image, 298 AAV8-injected pancreas exhibited comparable 299 green fluorescent brightness to that of transgenic 300 mice ( Figure 2B) Figure 3A, B). GFP production in pancreas 332 injected with AAV8 saturated at 10 10 VP, which 333 decreased with lower doses. ...
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Citations
... Previous experiments [32][33][34][35] suggested that AAV serotypes DJ and 8 would be the most appropriate for delivery into the pancreas. Eight-week-old Cas9-EGFP mice were subjected to tail vein injection of AAV-sgRNA-CDS of either serotype DJ or 8. ...
With the development of CRISPR/Cas9-mediated gene editing technologies, correction of disease-causing mutations has become possible. However, current gene correction strategies preclude mutation repair in post-mitotic cells of human tissues, and a unique repair strategy must be designed and tested for each and every mutation that may occur in a gene. We have developed a novel gene correction strategy, Co-opting Regulation Bypass Repair (CRBR), which can repair a spectrum of mutations in mitotic or post-mitotic cells and tissues. CRBR utilizes the non-homologous end-joining (NHEJ) pathway to insert a coding sequence (CDS) and transcription/translation terminators targeted upstream of any CDS mutation and downstream of the transcriptional promoter. CRBR results in simultaneous co-option of the endogenous regulatory region and bypass of the genetic defect. We validated the CRBR strategy for human gene therapy by rescuing a mouse model of Wolcott-Rallison syndrome (WRS) with permanent neonatal diabetes caused by either a large deletion or a nonsense mutation in the PERK (EIF2AK3) gene. Additionally, we integrated a CRBR GFP-terminator cassette downstream of the human insulin promoter in cadaver pancreatic islets of Langerhans which resulted in insulin promoter regulated expression of GFP, demonstrating the potential utility of CRBR in human tissue gene repair.
... We first transduced the mouse pancreas in vivo using the Adeno-Associated Viral (AAV) vector to express the NF-κB-responsive luciferase reporter, and then isolated the acinar cells and used only one adenoviral vector in vitro per well to perform the study. Compared to the adenovirus vector, the AAV vector has very negligible immunogenicity and is associated with undetectable inflammation [17][18][19][20]. Non-operated mice (male, 30-50 g, C57BL/6 from National Cancer Institute, Frederick, MD) were given AAV8.NF-κB.Luc 3e12 Viral Genome Particles (VGP) i.p. 2 wks prior to euthanasia. ...
Aim: To test the hypothesis that cooperative interactions between transcription factors Nuclear Factor-kappa B (NF-κB) and Activator Protein-1 (AP1) augment transcriptional activity in exocrine pancreatic cells.
... Previous experiments [32][33][34] suggested that AAV serotypes DJ and 8 would be the most appropriate for delivery into the pancreas. Eight-week old Cas9-EGFP mice were subjected to tail vein injection of AAV-sgRNA-CDS of either serotype DJ or 8. ...
With the development of CRISPR/Cas9-mediated gene editing technologies, correction of disease- causing mutations has become possible. However, current gene correction strategies preclude mutation repair in post-mitotic cells of human tissues, and a unique repair strategy must be designed and tested for each and every mutation that may occur in a gene. We have developed a novel gene correction strategy, Co-opting Regulation Bypass Repair (CRBR), which can repair a spectrum of mutations in mitotic or post-mitotic cells and tissues. CRBR utilizes the non-homologous end-joining (NHEJ) pathway to insert a coding sequence (CDS) and transcription/translation terminators targeted upstream of any CDS mutation and downstream of the transcriptional promoter. CRBR gene repair results in simultaneous co-option of the endogenous regulatory region and bypass of the genetic defect. We demonstrated the potential of CRBR strategy for human gene therapy by rescuing a mouse model of the Wolcott-Rallison syndrome (WRS) with permanent neonatal diabetes caused by either large deletion or nonsense mutation in the PERK (EIF2AK3) gene. Additionally, we expressed a GFP CDS-terminator cassette that was integrated downstream of the human insulin promoter in cadaver pancreatic islets of Langerhans which paves the way for autologous cell-tissue replacement therapy for gene repair in beta cells.
... Adenoassociated vectors (AAV) are emerging as a preferred vehicle for therapeutic cell engineering, 37 and while tested in experimental studies using human, porcine and rodent islets the relatively low transduction efficiency of AAV toward intact islets limits the utility of this gene engineering approach and could be improved. [38][39][40][41][42][43][44] To advance clinical translation, we developed an AAV-based protocol that achieved high-efficiency engineering of intact NPI (>70% cells transduced) and demonstrate that this can effectively deliver A20 to NPIs and suppress inflammation. ...
... We developed a novel NPI gene engineering strategy that combined a NPI differentiation protocol with a selected AAV serotype that routinely achieved > 70% gene transduction, a dramatic improvement from the ~ 20% transduction efficiency often reported. [39][40][41][42] We utilized a single-strand (ss) AAV vector to accommodate Each of these steps is influenced by the specific vector capsid used. ...
Background
Neonatal porcine islets (NPIs) can restore glucose control in mice, pigs, and non‐human primates, representing a potential abundant alternative islet supply for clinical beta cell replacement therapy. However, NPIs are vulnerable to inflammatory insults that could be overcome with genetic modifications. Here, we demonstrate in a series of proof‐of‐concept experiments the potential of the cytoplasmic ubiquitin‐editing protein A20, encoded by the TNFAIP3 gene, as an NPI cytoprotective gene.
Methods
We forced A20 expression in NPI grafts using a recombinant adenovirus 5 (Ad5) vector and looked for impact on TNF‐stimulated NF‐κB activation and NPI graft function. As adeno‐associated vectors (AAV) are clinically preferred vectors but exhibit poor transduction efficacy in NPIs, we next screened a series of AAV serotypes under different transduction protocols for their ability achieve high transduction efficiency and suppress NPI inflammation without impacting NPI maturation.
Results
Forcing the expression of A20 in NPI with Ad5 vector blocked NF‐κB activation by inhibiting IκBα phosphorylation and degradation, and reduced the induction of pro‐inflammatory genes Cxcl10 and Icam1. A20‐expressing NPIs also exhibited superior functional capacity when transplanted into diabetic immunodeficient recipient mice, evidenced by a more rapid return to euglycemia and improved GTT compared to unmodified NPI grafts. We found AAV2 combined with a 14‐day culture period maximized NPI transduction efficiency (>70% transduction rate), and suppressed NF‐κB‐dependent gene expression without adverse impact upon NPI maturation.
Conclusion
We report a new protocol that allows for high‐efficiency genetic modification of NPIs, which can be utilized to introduce candidate genes without the need for germline engineering. This approach would be suitable for preclinical and clinical testing of beneficial molecules. We also report for the first time that A20 is cytoprotective for NPI, such that A20 gene therapy could aid the clinical development of NPIs for beta cell replacement.
... 28,29 Ad vector-mediated pancreatic gene transfer is transient in nature because of potent host immune responses and vector-mediated cytotoxicity. [30][31][32] On the other hand, non-integrating AAV vectors are particularly promising in efficiently targeting the whole pancreas, [33][34][35] have the potential to target specific cell types in the pancreas [36][37][38] and pancreatic neoplasms, 39 and do not elicit humoral responses or alterations in pancreatic functions. [33][34][35]39,40 AAV vectors contain a single-stranded DNA (ssDNA) genome with a packaging capacity of $4.8 kb and are able to mediate long-term transgene expression because of their lack of pathogenicity and low immunogenicity. ...
... [30][31][32] On the other hand, non-integrating AAV vectors are particularly promising in efficiently targeting the whole pancreas, [33][34][35] have the potential to target specific cell types in the pancreas [36][37][38] and pancreatic neoplasms, 39 and do not elicit humoral responses or alterations in pancreatic functions. [33][34][35]39,40 AAV vectors contain a single-stranded DNA (ssDNA) genome with a packaging capacity of $4.8 kb and are able to mediate long-term transgene expression because of their lack of pathogenicity and low immunogenicity. [41][42][43] In addition, ssAAV vectors have been engineered to contain a double-stranded DNA (dsDNA) genome (hairpin) to circumvent the requirement for second-strand DNA synthesis, a requisite for transgene expression, and are self-complementary (sc) AAV viral vectors. ...
... 46 Determining an ideal delivery route with limited off-target effects is another critical component to target the pancreas efficiently. A wide range of gene delivery routes have been exploited, such as direct pancreatic injections, 30,35,47 intraperitoneal delivery, 39,48 systemic delivery, 49,50 in conjunction with clamped hepatic circulation, 33 celiac/hepatic artery delivery, 51 retrograde ductal delivery via pancreaticobiliary ductal infusion, 21,28,34,40 or catheterizing the cystic duct through the gallbladder/common bile duct. 25,33 Among several AAV serotypes tested, AAV8 and AAV9 have been shown to be well-suited for systemic delivery, 52,53 and AAV6 has been shown to transduce the normal pancreas very efficiently via retrograde pancreatic ductal delivery. ...
... 28,29 Ad vector-mediated pancreatic gene transfer is transient in nature because of potent host immune responses and vector-mediated cytotoxicity. [30][31][32] On the other hand, non-integrating AAV vectors are particularly promising in efficiently targeting the whole pancreas, [33][34][35] have the potential to target specific cell types in the pancreas [36][37][38] and pancreatic neoplasms, 39 and do not elicit humoral responses or alterations in pancreatic functions. [33][34][35]39,40 AAV vectors contain a single-stranded DNA (ssDNA) genome with a packaging capacity of $4.8 kb and are able to mediate long-term transgene expression because of their lack of pathogenicity and low immunogenicity. ...
... [30][31][32] On the other hand, non-integrating AAV vectors are particularly promising in efficiently targeting the whole pancreas, [33][34][35] have the potential to target specific cell types in the pancreas [36][37][38] and pancreatic neoplasms, 39 and do not elicit humoral responses or alterations in pancreatic functions. [33][34][35]39,40 AAV vectors contain a single-stranded DNA (ssDNA) genome with a packaging capacity of $4.8 kb and are able to mediate long-term transgene expression because of their lack of pathogenicity and low immunogenicity. [41][42][43] In addition, ssAAV vectors have been engineered to contain a double-stranded DNA (dsDNA) genome (hairpin) to circumvent the requirement for second-strand DNA synthesis, a requisite for transgene expression, and are self-complementary (sc) AAV viral vectors. ...
... 46 Determining an ideal delivery route with limited off-target effects is another critical component to target the pancreas efficiently. A wide range of gene delivery routes have been exploited, such as direct pancreatic injections, 30,35,47 intraperitoneal delivery, 39,48 systemic delivery, 49,50 in conjunction with clamped hepatic circulation, 33 celiac/hepatic artery delivery, 51 retrograde ductal delivery via pancreaticobiliary ductal infusion, 21,28,34,40 or catheterizing the cystic duct through the gallbladder/common bile duct. 25,33 Among several AAV serotypes tested, AAV8 and AAV9 have been shown to be well-suited for systemic delivery, 52,53 and AAV6 has been shown to transduce the normal pancreas very efficiently via retrograde pancreatic ductal delivery. ...
Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 10¹² viral genomes (vg). Intraductal delivery of 1 × 10¹¹ vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 10¹¹ vg. In a KrasG12D-driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.
... Fibroblast growth factor receptor, αvβ5 integrin, and hepatocyte growth factor receptor have been reported to work as co-factors [36][37][38]. Among the many types of AAV, single-stranded AAV, such as serotypes 8 (AAV-8) and 9 (AAV-9), and double-stranded AAV have been reported to have effective transduction of transgenes into the pancreas [39][40][41][42][43][44]. ...
Gene therapy that targets the pancreas and intestines with delivery systems using nano-sized carriers such as viral and non-viral vectors could improve the control of blood glucose levels, resulting in an improved prognosis for patients with diabetes mellitus. Allogenic pancreatic islet cell transplantations using such delivery systems have been developed as therapeutic options for diabetes mellitus. This review focuses on transgenes and islet cell delivery systems using nano-sized carriers for the treatment of diabetes mellitus.
... Robust transduction of both endocrine and exocrine pancreas was observed 2 weeks post-injection ( Figure 5A,B). However, the use of the ubiquitous CAG promoter also led to production of GFP in the liver and heart ( Figure 5C), in agreement with previous reports in other mouse strains [20,22,23]. To prevent transgene expression in liver and heart and restrict AAV8mediated IGF1 overexpression to pancreas, we took advantage of microRNAs (miRs) [26]. ...
Objective: Type 1 diabetes is characterized by autoimmune destruction of β-cells leading to severe insulin deficiency. Although many improvements have been made in recent years, exogenous insulin therapy is still imperfect; new therapeutic approaches, focusing on preserving/expanding β-cell mass and/or blocking the autoimmune process that destroys islets, should be developed. The main objective of this work was to test in non-obese diabetic (NOD) mice, which spontaneously develop autoimmune diabetes, the effects of local expression of Insulin-like growth factor 1 (IGF1), a potent mitogenic and pro-survival factor for β-cells with immunomodulatory properties.
Methods: Transgenic NOD mice overexpressing IGF1 specifically in β-cells (NOD-IGF1) were generated and phenotyped. In addition, miRT-containing, IGF1-encoding adeno-associated viruses (AAV) of serotype 8 (AAV8-IGF1-dmiRT) were produced and administered to 4- or 11-week-old non-transgenic NOD females through intraductal delivery. Several histological, immunological, and metabolic parameters were measured to monitor disease over a period of 28–30 weeks.
Results: In transgenic mice, local IGF1 expression led to long-term suppression of diabetes onset and robust protection of β-cell mass from the autoimmune insult. AAV-mediated pancreatic-specific overexpression of IGF1 in adult animals also dramatically reduced diabetes incidence, both when vectors were delivered before pathology onset or once insulitis was established. Transgenic NOD-IGF1 and AAV8-IGF1-dmiRT-treated NOD animals had much less islet infiltration than controls, preserved β-cell mass, and normal insulinemia. Transgenic and AAV-treated islets showed less expression of antigen-presenting molecules, inflammatory cytokines, and chemokines important for tissue-specific homing of effector T cells, suggesting IGF1 modulated islet autoimmunity in NOD mice.
Conclusions: Local expression of Igf1 by AAV-mediated gene transfer counteracts progression to diabetes in NOD mice. This study suggests a therapeutic strategy for autoimmune diabetes in humans.
... [15][16][17] Furthermore, AAV8 crosses vascular endothelial barriers more efficiently than other serotypes, resulting in efficient gene delivery to hepatic, cardiac, and skeletal muscle cells. 18,19 The first successful AAV8-mediated gene transfer has been achieved in patients with hemophilia B. 20 AAV8 also shows modest transduction of normal exocrine and endocrine pancreas when delivered systemically, 21,22 intrapancreatically, 23,24 intraductally, 22,25 or through the intrapancreatic vessels in animal models. 26 The remaining obstacles for pancreatic tropism includes, irrespective of the serotype employed, the persistence of vector dose-dependent immune response and the modest level of transduction efficacy. ...
... We also observed that i.p. injection of WT-AAV8 and Y447F+Y733F-AAV8 into 2-month-old FVB/C129 mice predominantly transduce exocrine pancreatic tissues; however, Image J analysis performed on the same samples showed 1-5% transduction efficiency of WT-AAV8 and Y447F+Y733F-AAV8 in the endocrine pancreas as previously reported. 23 We observed furthermore that the enhanced transduction efficiency of the double mutant AAV8 correlated well with the increased vector genome copy numbers in the pancreas (Fig. 1B, C). Y447F+Y733F-AAV8 showed a 6.87-fold higher genome copy number as compared with the WT-AAV8 (10,285 vs. 1,500 vector copies/ lg DNA respectively, n = 3 per group, p < 0.05) (Fig. 1C). ...
... 39,58 More specifically, the use of AAV8 has led to longterm expression and phenotypic correction of Factor IX in patients with hemophilia B. 58 Although AAV8 have been used in preclinical animal models for the potential treatment of PDAC, only a modest level of transduction efficiency in the pancreas has been achieved. [21][22][23][24][25] We reasoned that this drawback could potentially be overcome by the use of a new capsidoptimized AAV8 vector. In our efforts to identify the most efficient Y-F mutant AAV8 vector to target normal pancreas or PDAC, we investigated three AAV8 capsid mutants (Y733F, Y447F+Y733F, and Y275F+Y447F+Y733F) and compared their efficiency with the WT-AAV8. ...
Despite efforts to use adeno-associated viral (AAV) vector-mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F, and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to Kras<sup>G12D+/-</sup>, Kras<sup>G12D+/-</sup>/Pten and wild-type mice. The transduction efficiency of these vectors expressing the mCherry reporter gene was evaluated two weeks post administration in pancreas or PDAC, and correlated with viral genome copy numbers. Our comparative and quantitative analyses of the transduction profiles demonstrated that the Y to F double-mutant exhibited the highest mCherry expression in pancreatic tissues (range 45-70%) compared with WT-AAV8 (7%; p<0.01). We also detected a 7-fold higher level of vector genome copy numbers in normal pancreas following transduction with the double-mutant AAV8 compared to WT-AAV8 (10,285 vs. 1,500 vector copies/μg DNA respectively, p<0.05). In addition, we observed that intraperitoneal injection of the double-mutant AAV8 led to a 15-fold enhanced transduction efficiency as compared to WT-AAV8 in mouse PDAC, with a corresponding ~14-fold increase in vector genome copy numbers (26,575 vs. 2,165 copies/μg DNA respectively, p<0.05). These findings indicate that the Y447+Y733F-AAV8 leads to a significant enhancement of transduction efficiency in both normal and malignant pancreatic tissues, suggesting the potential use of this vector in targeting pancreatic diseases in general, and PDAC in particular.
... In particular, AAV8 has yielded high transduction efficiency for both exocrine acinar cells and endocrine islet cells. 160 The effects of different administration routes (intraperitoneal, intravenous and intraductal) and different AAV serotypes (AAV1, 2, 5, 6, and 8) have been investigated for gene transfer in mice. Intraductal administration of AAV6 and intravenous administration of AAV8 both have been shown to be good methods for delivering genes to b-cells in vivo. ...
As a promising strategy to aid or replace tissue/organ transplantation, gene delivery has been used for regenerative medicine applications to create or restore normal function at the cell and tissue levels. Gene delivery has been successfully performed ex vivo and in vivo in these applications. Excellent proliferation capabilities and differentiation potentials render certain cells as excellent candidates for ex vivo gene delivery for regenerative medicine applications, which is why multipotent and pluripotent cells have been intensely studied in this vein. In this review, gene delivery is discussed in detail, along with its applications to tissue engineering and regenerative medicine. A definition of a stem cell is compared to a definition of a stem property, and both provide the foundation for an in-depth look at gene delivery investigations from a germ lineage angle.
