Candidate Vectors Research Articles

Co-overexpression of TGF-β and SOX9 via rAAV gene transfer modulates the metabolic and chondrogenic activities of human bone marrow-derived mesenchymal stem cells.

BackgroundArticular cartilage has a limited potential for self-healing. Transplantation of genetically modified progenitor cells like bone marrow-derived mesenchymal stem cells (MSCs) is an attractive strategy to improve the intrinsic repair capacities of damaged articular cartilage.MethodsIn this study, we examined the potential benefits of co-overexpressing the pleiotropic transformation growth factor beta (TGF-β) with the cartilage-specific transcription factor SOX9 via gene transfer with recombinant adeno-associated virus (rAAV) vectors upon the biological activities of human MSCs (hMSCs). Freshly isolated hMSCs were transduced over time with separate rAAV vectors carrying either TGF-β or sox9 in chondrogenically-induced aggregate cultures to evaluate the efficacy and duration of transgene expression and to monitor the effects of rAAV-mediated genetic modification upon the cellular activities (proliferation, matrix synthesis) and chondrogenic differentiation potency compared with control conditions (lacZ treatment, sequential transductions).ResultsSignificant, prolonged TGF-β/sox9 co-overexpression was achieved in chondrogenically-induced hMSCs upon co-transduction via rAAV for up to 21 days, leading to enhanced proliferative, biosynthetic, and chondrogenic activities relative to control treatments, especially when co-applying the candidate vectors at the highest vector doses tested. Optimal co-administration of TGF-β with sox9 also advantageously reduced hypertrophic differentiation of the cells in the conditions applied here.ConclusionThe present findings demonstrate the possibility of modifying MSCs by combined therapeutic gene transfer as potent future strategies for implantation in clinically relevant animal models of cartilage defects in vivo.

High Prevalence of West Nile Virus in Domestic Birds and Detection in 2 New Mosquito Species in Madagascar.

West Nile virus is an arthropod-borne zoonosis transmitted by a large number of mosquito species, and birds play a key role as reservoir of the virus. Its distribution is largely widespread over Africa, Asia, the Americas and Europe. Since 1978, it has frequently been reported in Madagascar. Studies described a high seroprevalence level of the virus in humans in different areas of the island and a human fatal case of WNV infection was reported in 2011. Despite these reports, the epidemiology of WNV in Madagascar, in particular, viral circulation remains unclear. To explore the transmission of WNV in two rural human populations of Madagascar, we investigated local mosquitoes and poultry for evidence of current infections, and determined seroprevalence of candidate sentinel species among the local poultry. These 2 areas are close to lakes where domestic birds, migratory wild birds and humans coexist. Serological analysis revealed WNV antibodies in domestic birds (duck, chicken, goose, turkey and guinea fowl) sampled in both districts (Antsalova 29.4% and Mitsinjo 16.7%). West Nile virus nucleic acid was detected in one chicken and in 8 pools of mosquitoes including 2 mosquito species (Aedeomyia madagascarica and Anopheles pauliani) that have not been previously described as candidate vectors for WNV. Molecular analysis of WNV isolates showed that all viruses detected were part of the lineage 2 that is mainly distributed in Africa, and were most closely matched by the previous Malagasy strains isolated in 1988. Our study showed that WNV circulates in Madagascar amongst domestic birds and mosquitoes, and highlights the utility of poultry as a surveillance tool to detect WNV transmission in a peri-domestic setting.

Review of West Nile virus circulation and outbreak risk in Madagascar: Entomological and ornithological perspectives.

West Nile fever (WNF) is a zoonotic disease, occurring nearly globally. In Madagascar, West Nile virus (WNV) was first detected in 1978 from wild birds and the virus is currently distributed across the island, but no epidemic or epizootic period has been recorded. One fatal human case of WNV infection was reported in 2011, suggesting a “tip of the iceberg” phenomenon of a possible WNF epidemic/epizootic on the island. The main objective of this literature-based survey is to review patterns of WNV circulation in Madagascar from the entomological and ornithological points of view. Among the 235 mosquito species described from Madagascar, 29 species are widely associated with WNV infection; 16 of them are found naturally infected with WNV on the island and categorized into major, candidate, and potential vectors of WNV according to their vector capacity. This study upholds the hypothesis that WNV enzooticity is independent of annual movements of migratory birds passing through Madagascar. Moreover, the lack of regular migratory bird flux between Africa and Madagascar would reduce the probability of transmission and the subsequent reintroduction of the virus into locally occurring mosquito species. Given that Palearctic migratory birds are strongly implicated in the transmission of WNV, we highlight notable differences in the movements and species diversity of these birds in Madagascar as compared to eastern and northern Africa. Risk factors from this two-pronged approach are presented for the emergence of WNF outbreak.

A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform

Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 1014 rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 105 vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production.

Effective mRNA Inhibition in PANC-1 Cells in Vitro Mediated via an mPEG-SeSe-PEI Delivery System.

RNA interference (RNAi)-mediated gene therapy is a promising approach to cure various diseases. However, developing an effective, safe, specific RNAi delivery system remains a major challenge. In this study, a novel redox-responsive polyetherimide (PEI)-based nanovector, mPEG-SeSe-PEI, was developed and its efficacy evaluated. We prepared three mPEG-SeSe-PEI vector candidates for small interfering glyceraldehyde-3-phosphate dehydrogenase (siGADPH) and determined their physiochemical properties and transfection efficiency using flow cytometry and PEG11.6-SeSe-PEI polymer. We investigated the silencing efficacy of GADPH mRNA expression in PANC-1 cells and observed that PEG11.6-SeSe-PEI/siGADPH (N/P ratio=10) polyplexes possessed the appropriate size and zeta-potential and exhibited excellent in vitro gene silencing effects with the least cytotoxicity in PANC-1 cells. In conclusion, we present PEG11.6-SeSe-PEI as a potential therapeutic gene delivery system for small interfering RNA (siRNA).

GNSS Single Frequency, Single Epoch Reliable Attitude Determination Method with Baseline Vector Constraint.

For Global Navigation Satellite System (GNSS) single frequency, single epoch attitude determination, this paper proposes a new reliable method with baseline vector constraint. First, prior knowledge of baseline length, heading, and pitch obtained from other navigation equipment or sensors are used to reconstruct objective function rigorously. Then, searching strategy is improved. It substitutes gradually Enlarged ellipsoidal search space for non-ellipsoidal search space to ensure correct ambiguity candidates are within it and make the searching process directly be carried out by least squares ambiguity decorrelation algorithm (LAMBDA) method. For all vector candidates, some ones are further eliminated by derived approximate inequality, which accelerates the searching process. Experimental results show that compared to traditional method with only baseline length constraint, this new method can utilize a priori baseline three-dimensional knowledge to fix ambiguity reliably and achieve a high success rate. Experimental tests also verify it is not very sensitive to baseline vector error and can perform robustly when angular error is not great.

Factors determining the stability, size distribution, and cellular accumulation of small, monodisperse chitosan nanoparticles as candidate vectors for anticancer drug delivery: application to the passive encapsulation of [(14)C]-doxorubicin.

Development of parameters for the fabrication of nanosized vectors is pivotal for its successful administration in therapeutic applications. In this study, homogeneously distributed chitosan nanoparticles (CNPs) with diameters as small as 62 nm and a polydispersity index (PDI) of 0.15 were synthesized and purified using a simple, robust method that was highly reproducible. Nanoparticles were synthesized using modified ionic gelation of the chitosan polymer with sodium tripolyphosphate. Using this method, larger aggregates were mechanically isolated from single particles in the nanoparticle population by selective efficient centrifugation. The presence of disaggregated monodisperse nanoparticles was confirmed using atomic force microscopy. Factors such as anions, pH, and concentration were found to affect the size and stability of nanoparticles directly. The smallest nanoparticle population was ∼62 nm in hydrodynamic size, with a low PDI of 0.15, indicating high particle homogeneity. CNPs were highly stable and retained their monodisperse morphology in serum-supplemented media in cell culture conditions for up to 72 hours, before slowly degrading over 6 days. Cell viability assays demonstrated that cells remained viable following a 72-hour exposure to 1 mg/mL CNPs, suggesting that the nanoparticles are well tolerated and highly suited for biomedical applications. Cellular uptake studies using fluorescein isothiocyanate-labeled CNPs showed that cancer cells readily accumulate the nanoparticles 30 minutes posttreatment and that nanoparticles persisted within cells for up to 24 hours posttreatment. As a proof of principle for use in anticancer therapeutic applications, a [14C]-radiolabeled form of the anticancer agent doxorubicin was efficiently encapsulated within the CNP, confirming the feasibility of using this system as a drug delivery vector.

Efficient induction of cross-presentating human B cell by transduction with human adenovirus type 7 vector

Although human autologous B cells represent a promising alternative to dendritic cells (DCs) for easy large-scale preparation, the naive human B cells are always poor at antigen presentation. The safe and effective usage record of human adenovirus type 7 (HAdV7) live vaccines makes it attractive as a promising vaccine vector candidate. To investigate whether HAdV7 vector could be used to induce the human B cells cross-presentation, in the present study, we constructed the E3-defective recombinant HAdV7 vector encoding green fluorescent protein (GFP) and carcinoembryonic antigen (CEA). We demonstrated that naive human B cells can efficiently be transduced, and that the MAPKs/NF-κB pathway can be activated by recombinant HAdV7. We proved that cytokine TNF-α, IL-6 and IL-10, surface molecule MHC class I and the CD86, antigen-processing machinery (APM) compounds ERp57, TAP-1, and TAP-2. were upregulated in HAdV7 transduced human B cells. We also found that CEA-specific IFNγ expression, degranulation, and in vitro and ex vivo cytotoxicities are induced in autologous CD8+ T cells presensitized by HAd7CEA modified human B cells. Meanwhile, our evidences clearly show that Toll-like receptors 9 (TLR9) antagonist IRS 869 significantly eliminated most of the HAdV7 initiated B cell activation and CD8+ T cells response, supporting the role and contribution of TLR9 signaling in HAdV7 induced human B cell cross-presentation. Besides a better understanding of the interactions between recombinant HAdV7 and human naive B cells, to our knowledge, the present study provides the first evidence to support the use of HAdV7-modified B cells as a vehicle for vaccines and immunotherapy.

Analysis of T cell responses to chimpanzee adenovirus vectors encoding HIV gag–pol–nef antigen

Adenoviruses have been shown to be both immunogenic and efficient at presenting HIV proteins but recent trials have suggested that they may play a role in increasing the risk of HIV acquisition. This risk may be associated with the presence of pre-existing immunity to the viral vectors. Chimpanzee adenoviruses (chAd) have low seroprevalence in human populations and so reduce this risk. ChAd3 and chAd63 were used to deliver an HIV gag, pol and nef transgene. ELISpot analysis of T cell responses in mice showed that both chAd vectors were able to induce an immune response to Gag and Pol peptides but that only the chAd3 vector induced responses to Nef peptides. Although the route of injection did not influence the magnitude of immune responses to either chAd vector, the dose of vector did. Taken together these results demonstrate that chimpanzee adenoviruses are suitable vector candidates for the delivery of HIV proteins and could be used for an HIV vaccine and furthermore the chAd3 vector produces a broader response to the HIV transgene.

Bio-inspired polymer envelopes around adenoviral vectors to reduce immunogenicity and improve in vivo kinetics

Adenoviral vectors have attracted substantial interest for systemic tumor gene therapy, but further work is needed to reduce their immunogenicity and alter their biodistribution before they can be used in the clinic. Here we describe a bio-inspired, cleavable PEGylated β-cyclodextrin-polyethyleneimine conjugate (CDPCP) that spontaneously coats adenovirus in solution. This cleavable PEG coating reduces the innate and adaptive immunogenicity of adenovirus particles, as well as improves their biodistribution away from the liver and into the tumor. Insertion of a matrix metalloproteinase substrate sequence into the conjugate allows PEG cleavage at the tumor site, simultaneously reducing liver biodistribution and increasing transgene expression in tumors, thereby avoiding the “PEG dilemma”. Cationic β-cyclodextrin–PEI not only provides electrostatic attraction to promote envelope attachment to the viral capsid, but it also improves vector internalization and transduction after PEG cleavage. These results suggest that CDPCP may help expand the use of adenoviral vectors in cancer gene therapy. Statement of significanceThe synthesized β-cyclodextrin–PEI-MMP-cleavable-PEG polymer (CDPCP), held great potential for gene therapy when applied for adenovirus coating. The β-cyclodextrin–PEI provided a powerful electrostatic attraction to attach the whole polymer onto the viral capsid, while the MMPs-cleavable PEG reduced innate and adaptive immunogenicity and improved the biodistribution of adenovirus vectors due to the tumor-specific enzyme triggered PEG cleavage. More importantly, an ingenious cooperation between the two components could solve the PEG dilemma. The CDPCP/Ad complexes exhibited a comprehensive and valued profile to be a candidate vector for future tumor gene therapy, we believe the current investigation on this kind of biomaterial may be of particular interest to the readership of Acta biomaterialia.

Estimating dynamic discrete-choice games of incomplete information

We investigate the estimation of models of dynamic discrete-choice games of incomplete information, formulating the maximum-likelihood estimation exercise as a constrained optimization problem that can be solved using state-of-the-art constrained optimization solvers. Under the assumption that only one equilibrium is played in the data, our approach avoids repeatedly solving the dynamic game or finding all equilibria for each candidate vector of the structural parameters. We conduct Monte Carlo experiments to investigate the numerical performance and finite-sample properties of the constrained optimization approach for computing the maximum-likelihood estimator, the two-step pseudo-maximum-likelihood estimator, and the nested pseudo-likelihood estimator, implemented by both the nested pseudo-likelihood algorithm and a modified nested pseudo-likelihood algorithm.

An improved region growing algorithm for phase correction in MRI.

To develop an improved region-growing algorithm for phase correction in MRI. Phase correction in MRI can sometimes be formulated as selecting a vector for each pixel of an image from two candidate vectors so that the orientation of the output is spatially smooth. Existing algorithms may run into difficulty in the presence of high noise, artifacts, or spatially isolated objects. In this study, we developed an improved region-growing algorithm to include the following novel and salient features: 1) automated quality guidance for determining the sequence of region growing, 2) joint consideration of two candidate vectors in selecting the output vector, and 3) automated segmentation during region growing for handling spatially isolated objects. The phase correction algorithm was tested in different body parts of five healthy volunteers at 3.0T and of one healthy volunteer at 1.5T for two-point Dixon water and fat imaging with flexible echo times. The algorithm achieved successful phase correction in all the data sets tested, providing improvement in areas of known difficulty, when compared with an algorithm lacking the new features. The improved region-growing algorithm can be used for reliable and robust phase correction even when regions of high noise, artifacts, or spatially isolated objects are present in an image. Magn Reson Med 76:519-529, 2016. © 2015 Wiley Periodicals, Inc.

Common‐mode voltage mitigation with a predictive control method considering dead time effects of three‐phase voltage source inverters

This study proposes a technique to mitigate common-mode voltages with dead time effects taken into account in three-phase voltage source inverters (VSIs), on the basis of finite-set model predictive control methods. The effects of dead time on common-mode voltage in three-phase VSIs controlled by the model predictive control method are investigated. On the basis of the investigation, a common-mode voltage reduction method is proposed, which is not affected by the inevitable dead time of the VSIs. The proposed method pre-excludes, from the candidates for future vectors, those voltage vectors which can increase the common-mode voltage during the dead time. In addition, when zero vectors are chosen as an optimal vector from the standpoints of current errors, each zero vector is replaced with two non-zero vectors in opposite directions, so as not to deteriorate current performance by simply excluding the zero vectors. On the basis of the proposed pre-selective technique, the proposed method can restrict the common-mode voltage within ± V dc / 6 of the three-phase VSIs, whereas the common-mode voltages of the conventional method vary within ± V dc /2. Simulation and experimental results are presented to verify the effectiveness of the proposed method.

Evaluation of Readministration of a Recombinant Adeno-Associated Virus Vector Expressing Acid Alpha-Glucosidase in Pompe Disease: Preclinical to Clinical Planning.

A recombinant serotype 9 adeno-associated virus (rAAV9) vector carrying a transgene that expresses codon-optimized human acid alpha-glucosidase (hGAA, or GAA) driven by a human desmin (DES) promoter (i.e., rAAV9-DES-hGAA) has been generated as a clinical candidate vector for Pompe disease. The rAAV9-DES-hGAA vector is being developed as a treatment for both early- and late-onset Pompe disease, in which patients lack sufficient lysosomal alpha-glucosidase leading to glycogen accumulation. In young patients, the therapy may need to be readministered after a period of time to maintain therapeutic levels of GAA. Administration of AAV-based gene therapies is commonly associated with the production of neutralizing antibodies that may reduce the effectiveness of the vector, especially if readministration is required. Previous studies have demonstrated the ability of rAAV9-DES-hGAA to correct cardiac and skeletal muscle pathology in Gaa(-/-) mice, an animal model of Pompe disease. This article describes the IND-enabling preclinical studies supporting the program for a phase I/II clinical trial in adult patients with Pompe. These studies were designed to evaluate the toxicology, biodistribution, and potential for readministration of rAAV9-DES-hGAA injected intramuscularly into the tibialis anterior muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with late-onset Pompe disease will be enrolled. The goal of the immune modulation strategy is to ablate B-cells before the initial exposure of the study agent in one leg and the subsequent exposure of the same vector to the contralateral leg four months after initial dosing. The dosing of the active agent is accompanied by a control injection of excipient dosing in the contralateral leg to allow for blinding and randomization of dosing, which may also strengthen the evidence generated from gene therapy studies in the future. Patients will act as their own controls. Repeated measures, at baseline and during the three months following each dosing will assess the safety, biochemical, and functional impact of the vector.

Synthesis and evaluation of novel chitosan derivatives for gene delivery

A series of novel water soluble chitosan derivatives as gene vectors was synthesized. The delivery systems were tested for their ability to form complexes with plasmid DNA by utilizing agarose gel electrophoresis, particle size analysis, zeta potential measurement and scanning electron microscopy. Furthermore, cytotoxicity of chitosan derivatives and transfection efficiency of polyplexes were evaluated in vitro. The experimental results showed that the novel chitosan derivatives had lower cytotoxicity, good DNA condensation, and higher transfection efficiencies compared to chitosan in both 293T and HeLa cell lines. It was indicated that these chitosan derivatives were promising candidates for non-viral gene vectors

Stability of the B. abortus S19 vaccine strain with a eukaryotic expression plasmid encoding the G glycoprotein from the rabies virus

Veterinaria México OA ISSN: 2448-6760Cite this as:Pazos-Salazar NG, Benitez-Serrano JC, Calderón-Chamorro JL, Hernández-Castro R, Díaz-Aparicio E, Aguilar-Setién JA. Stability of the B. abortus S19 vaccine strain with a eukaryotic expression plasmid encoding the G glycoprotein from the rabies virus. Veterinaria México OA. 2015;2(2). doi: 10.21753/vmoa.2.2.347.Brucella abortus S19 is an intracellular vaccine strain against bovine brucellosis. Rabies is a lethal disease in cattle. Plasmids encoding the G glycoprotein from the rabies virus induce a protective immune response in different animal species. A vector called pBBR4-CMV-Ggp-SV40+, which encodes the G gene, regulated by the cytomegalovirus eukaryotic expression promoter, and which can be used to transform the B. abortus S19 vaccine strain, was constructed. The stability of the transformant strain was tested both in vitro and in vivo. In the in vitro assays, B. abortus S19 pBBR4-CMV-Ggp-SV40+ was grown for 5 sequential passages, and for the in vivo assays, female BALB/c mice were infected. Colony-forming unit counting and plasmid identification were performed in each passage and in the spleens at 7 days post-infection. To test the plasmid stability in the strain, all parameters were determined with and without antibiotic. The bacterial concentration was lower with antibiotic than without it, but the bacterial growth was more homogeneous. The plasmid was identified in antibiotic- and non-antibiotic-treated isolated colonies under both in vitro and in vivo conditions. The plasmid construct was also transfected into BHK-21 cells, which express the G glycoprotein. The B. abortus S19 pBBR4-CMV-Ggp-SV40+ strain showed stability, representing a suitable candidate vector for developing a bivalent vaccine against brucellosis and rabies. This is the first time that a Brucella species has been transformed with a eukaryotic expression plasmid.Figure 1. Plasmid pBBR4-CMV-Ggp-SV40+ construction diagram.

Stability of the B. abortus S19 vaccine strain with a eukaryotic expression plasmid encoding the G glycoprotein from the rabies virus

Brucella abortus S19 is an intracellular vaccine strain against bovine brucellosis. Rabies is a lethal disease in cattle. Plasmids encoding the G glycoprotein from the rabies virus induce a protective immune response in different animal species. A vector called pBBR4-CMV-Ggp-SV40+, which encodes the G gene, regulated by the cytomegalovirus eukaryotic expression promoter, and which can be used to transform the B. abortus S19 vaccine strain, was constructed. The stability of the transformant strain was tested both in vitro and in vivo. In the in vitro assays, B. abortus S19 pBBR4-CMV-Ggp-SV40+ was grown for 5 sequential passages, and for the in vivo assays, female BALB/c mice were infected. Colony-forming unit counting and plasmid identification were performed in each passage and in the spleens at 7 days post-infection. To test the plasmid stability in the strain, all parameters were determined with and without antibiotic. The bacterial concentration was lower with antibiotic than without it, but the bacterial growth was more homogeneous. The plasmid was identified in antibiotic- and non-antibiotic-treated isolated colonies under both in vitro and in vivo conditions. The plasmid construct was also transfected into BHK-21 cells, which express the G glycoprotein. The B. abortus S19 pBBR4-CMV-Ggp-SV40+ strain showed stability, representing a suitable candidate vector for developing a bivalent vaccine against brucellosis and rabies. This is the first time that a Brucella species has been transformed with a eukaryotic expression plasmid.

MSE-Based Hybrid RF/Baseband Processing for Millimeter-Wave Communication Systems in MIMO Interference Channels

We consider the design of a hybrid multiple-input multiple-output (MIMO) processor consisting of a radio frequency (RF) beamformer and a baseband MIMO processor for millimeter-wave communications over multiuser interference channels. Sparse approximation problems are formulated to design hybrid MIMO processors approximating the minimum-mean-square-error transmit/receive processors in MIMO interference channels. They are solved by orthogonal-matching-pursuit-based algorithms that successively select RF beamforming vectors from a set of candidate vectors and optimize the corresponding baseband processor in the least squares sense. It is shown that various beamformers can be designed by considering different types of candidate vector sets. Simulation results demonstrate the advantage of the proposed design over the conventional method that designs the baseband processor after steering the RF beams.

262. Directed Evolution of Improved AAV Capsids for the Ideal Human Liver Vector–Can Human Liver Tropism and Human Immune Evasion Be Achieved?

Research towards improved adeno-associated virus (AAV) delivery vectors for use in treating human liver diseases continues to be dominated by efforts to characterize potential candidate vectors in mice which often can't recapitulate either the human liver tropism/transduction profiles, or the specifics of the human immune response to these vectors. Research from our lab has shown that despite the impressive hepatocyte transduction levels by AAV-8 in mouse liver, AAV-8 does not functionally transduce human hepatocytes in xenografted humanized liver mouse models to levels of therapeutic relevance, possibly explaining the lower than expected FIX expression levels from the recent AAV-8 hemophilia clinical trial. Efforts to evolve novel capsid variants with improved human hepatocyte tropism were successful with the generation of AAV-LK03, yet limitations to clinical translation with many newly evolved serotypes remains due to high levels of pre-existing neutralizing antibodies that may cross-react and neutralize transduction. For this reason we sought to evolve improved hepatotropic variants–again in humanized liver mice–that not only retained robust human liver tropism, but also had been evolved for resistance to pre-existing neutralizing antibodies in the human population to create the ideal liver vector for use in the clinic. We utilized wild-type replicating AAV libraries of 10e5 variants via DNA shuffling of eleven different parental AAV capsids. Our libraries selectively replicate in human cells when co-administered with wild-type adenovirus, making chimeric humanized liver mice an excellent tool to allow for selection of capsids with tropism for human liver. Screens were carried out for five rounds of selection and rather than waiting for the screen to go to completion, we ended the screen with some library diversity remaining for use in a subsequent screen for neutralizing antibody evasion. All variants from round five of the human liver screen were carried forward and screened for two additional rounds for their ability to resist binding to pooled human immunoglobulins in IgG immunocapture assays. The top 100 highly selected variants were sequenced and vectorized into CAG-driven GFP preparations in seven pools based on capsid relatedness at the amino acid level. Each pool was then resubjected to additional pooled human immunoglobulin screening and the best pool was chosen. The top seven candidates from this pool were tested alongside control serotypes that represent the extremes for humoral neutralization: highest neutralization (AAV-2), lowest neutralization (AAV-DJ), as well as LK03 that falls in between. One variant from this highly selected pool has neutralization profiles on par with AAV-DJ and further characterizations are underway in humanized liver mice in vivo and various human hepatoma lines in vitro to determine if both desired characteristics were robustly retained—human liver tropism and human immune evasion.

194. Lymphatic Transport Mediates Systemic Leakage of Peripherally Administered AAV9 Vectors

Adeno-associated viruses (AAV) have emerged as promising vector candidates for gene therapy of central nervous system (CNS) disorders. Preclinical studies in rodents as well as large animal models such as cats and non-human primates have shown that AAV9 mediates robust and widespread gene expression in the brain and spinal cord. In particular, after intracranial or intrathecal administration, leakage into the systemic circulation and accumulation of vector genomes in off-target organs such as the liver or spleen presents a potential safety concern. Here, we demonstrate that intrathecal administration of AAV9 transduces not only systemic and peripheral tissues, but also the peripheral lymph nodes. Significantly higher vector genome copy numbers were recovered from both systemic and lymphoid organs. Furthermore, AAV9 vectors are found specifically in the draining popliteal lymph nodes following hind footpad injection, an established model for studying lymphatic transport. Vector genomes from the blood circulation were recovered as early as 1 hour postadministration through intrathecal or footpad routes suggesting rapid transport. Similar observations were made in case of AAV8 vectors. In contrast, AAV2 vectors injected through either route were only recovered at the site of injection. Mutation of heparin binding residues on the AAV2 capsid mediated robust spread of vectors through the lymphatic drainage and systemic leakage. Together, these observations suggest that lymphatic vessels and lymph nodes are likely conduits for the systemic spread of AAV9 vectors following intrathecal administration. Further investigation is required to study the mechanisms that determine lymphatic transport of AAV9 vectors. These findings are important towards establishing the safety of gene therapy-based approaches to treat neurological diseases.