scholarly journals Overcoming Immunological Challenges Limiting Capsid-Mediated Gene Therapy With Machine Learning

2021 ◽  
Vol 12 ◽  
Author(s):  
Anna Z. Wec ◽  
Kathy S. Lin ◽  
Jamie C. Kwasnieski ◽  
Sam Sinai ◽  
Jeff Gerold ◽  
...  
Keyword(s):  
Machine Learning ◽  
Gene Therapy ◽  
Immune Responses ◽  
High Throughput ◽  
Transduction Efficiency ◽  
Adeno Associated Virus ◽  
Viral Immunology ◽  
Sequencing Technologies ◽  
Packaging Efficiency ◽  
New Generation

A key hurdle to making adeno-associated virus (AAV) capsid mediated gene therapy broadly beneficial to all patients is overcoming pre-existing and therapy-induced immune responses to these vectors. Recent advances in high-throughput DNA synthesis, multiplexing and sequencing technologies have accelerated engineering of improved capsid properties such as production yield, packaging efficiency, biodistribution and transduction efficiency. Here we outline how machine learning, advances in viral immunology, and high-throughput measurements can enable engineering of a new generation of de-immunized capsids beyond the antigenic landscape of natural AAVs, towards expanding the therapeutic reach of gene therapy.

scholarly journals Adenosine-to-inosine RNA editing may be implicated in human pathogenesis

2019 ◽  
pp. 22-25
Author(s):  
AA Kliuchnikova ◽  
SA Moshkovskii
Keyword(s):  
Immune Responses ◽  
Rna Editing ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Common Mechanism ◽  
Adenosine Deaminases ◽  
Human Transcriptome ◽  
Sequencing Technologies

Adenosine-to-inosine (A-to-I) RNA editing is a common mechanism of post-transcriptional modification in many metazoans including vertebrates; the process is catalyzed by adenosine deaminases acting on RNA (ADARs). Using high-throughput sequencing technologies resulted in finding thousands of RNA editing sites throughout the human transcriptome however, their functions are still poorly understood. The aim of this brief review is to draw attention of clinicians and biomedical researchers to ADAR-mediated RNA editing phenomenon and its possible implication in development of neuropathologies, antiviral immune responses and cancer.

scholarly journals The 37/67-Kilodalton Laminin Receptor Is a Receptor for Adeno-Associated Virus Serotypes 8, 2, 3, and 9

2006 ◽  
Vol 80 (19) ◽  
pp. 9831-9836 ◽  
Author(s):  
Bassel Akache ◽  
Dirk Grimm ◽  
Kusum Pandey ◽  
Stephen R. Yant ◽  
Hui Xu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cultured Cells ◽  
Human Gene ◽  
Laminin Receptor ◽  
Transduction Efficiency ◽  
Adeno Associated Virus ◽  
Virus Serotype ◽  
Tumor Gene

ABSTRACT Adeno-associated virus serotype 8 (AAV8) is currently emerging as a powerful gene transfer vector, owing to its capability to efficiently transduce many different tissues in vivo. While this is believed to be in part due to its ability to uncoat more readily than other AAV serotypes such as AAV2, understanding all the processes behind AAV8 transduction is important for its application and optimal use in human gene therapy. Here, we provide the first report of a cellular receptor for AAV8, the 37/67-kDa laminin receptor (LamR). We document binding of LamR to AAV8 capsid proteins and intact virions in vitro and demonstrate its contribution to AAV8 transduction of cultured cells and mouse liver in vivo. We also show that LamR plays a role in transduction by three other closely related serotypes (AAV2, -3, and -9). Sequence and deletion analysis allowed us to map LamR binding to two protein subdomains predicted to be exposed on the AAV capsid exterior. Use of LamR, which is constitutively expressed in many clinically relevant tissues and is overexpressed in numerous cancers, provides a molecular explanation for AAV8's broad tissue tropism. Along with its robust transduction efficiency, our findings support the continued development of AAV8-based vectors for clinical applications in humans, especially for tumor gene therapy.

scholarly journals Identification and Validation of Small Molecules That Enhance Recombinant Adeno-associated Virus Transduction following High-Throughput Screens

2016 ◽  
Vol 90 (16) ◽  
pp. 7019-7031 ◽  
Author(s):  
Sarah C. Nicolson ◽  
Chengwen Li ◽  
Matthew L. Hirsch ◽  
Vincent Setola ◽  
R. Jude Samulski
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
High Throughput ◽  
Adeno Associated Virus ◽  
Virus Gene ◽  
Diploid Cells ◽  
High Throughput Screens ◽  
Group 1

ABSTRACTWhile the recent success of adeno-associated virus (AAV)-mediated gene therapy in clinical trials is promising, challenges still face the widespread applicability of recombinant AAV(rAAV). A major goal is to enhance the transduction efficiency of vectors in order to achieve therapeutic levels of gene expression at a vector dose that is below the immunological response threshold. In an attempt to identify novel compounds that enhance rAAV transduction, we performed two high-throughput screens comprising 2,396 compounds. We identified 13 compounds that were capable of enhancing transduction, of which 12 demonstrated vector-specific effects and 1 could also enhance vector-independent transgene expression. Many of these compounds had similar properties and could be categorized into five groups: epipodophyllotoxins (group 1), inducers of DNA damage (group 2), effectors of epigenetic modification (group 3), anthracyclines (group 4), and proteasome inhibitors (group 5). We optimized dosing for the identified compounds in several immortalized human cell lines as well as normal diploid cells. We found that the group 1 epipodophyllotoxins (teniposide and etoposide) consistently produced the greatest transduction enhancement. We also explored transduction enhancement among single-stranded, self-complementary, and fragment vectors and found that the compounds could impact fragmented rAAV2 transduction to an even greater extent than single-stranded vectors.In vivoanalysis of rAAV2 and all of the clinically relevant compounds revealed that, consistent with ourin vitroresults, teniposide exhibited the greatest level of transduction enhancement. Finally, we explored the capability of teniposide to enhance transduction of fragment vectorsin vivousing an AAV8 capsid that is known to exhibit robust liver tropism. Consistent with ourin vitroresults, teniposide coadministration greatly enhanced fragmented rAAV8 transduction at 48 h and 8 days. This study provides a foundation based on the rAAV small-molecule screen methodology, which is ideally used for more-diverse libraries of compounds that can be tested for potentiating rAAV transduction.IMPORTANCEThis study seeks to enhance the capability of adeno-associated viral vectors for therapeutic gene delivery applicable to the treatment of diverse diseases. To do this, a comprehensive panel of FDA-approved drugs were tested in human cells and in animal models to determine if they increased adeno-associated virus gene delivery. The results demonstrate that particular groups of drugs enhance adeno-associated virus gene delivery by unknown mechanisms. In particular, the enhancement of gene delivery was approximately 50 to 100 times better with than without teniposide, a compound that is also used as chemotherapy for cancer. Collectively, these results highlight the potential for FDA-approved drug enhancement of adeno-associated virus gene therapy, which could result in safe and effective treatments for diverse acquired or genetic diseases.

scholarly journals Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation

2001 ◽  
Vol 75 (16) ◽  
pp. 7662-7671 ◽  
Author(s):  
Dongsheng Duan ◽  
Ziying Yan ◽  
Yongping Yue ◽  
Wei Ding ◽  
John F. Engelhardt
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Gene Delivery ◽  
Muscle Cells ◽  
Therapeutic Benefit ◽  
Transduction Efficiency ◽  
Great Promise ◽  
Adeno Associated Virus ◽  
Muscle Gene ◽  
Duchenne's Muscular Dystrophy

ABSTRACT Adeno-associated virus (AAV)-based muscle gene therapy has achieved tremendous success in numerous animal models of human diseases. Recent clinical trials with this vector have also demonstrated great promise. However, to achieve therapeutic benefit in patients, large inocula of virus will likely be necessary to establish the required level of transgene expression. For these reasons, efforts aimed at increasing the efficacy of AAV-mediated gene delivery to muscle have the potential for improving the safety and therapeutic benefit in clinical trials. In the present study, we compared the efficiency of gene delivery to mouse muscle cells for recombinant AAV type 2 (rAAV-2) and rAAV-2cap5 (AAV-2 genomes pseudo-packaged into AAV-5 capsids). Despite similar levels of transduction by these two vectors in undifferentiated myoblasts, pseudotyped rAAV-2cap5 demonstrated dramatically enhanced transduction in differentiated myocytes in vitro (>500-fold) and in skeletal muscle in vivo (>200-fold) compared to rAAV-2. Serotype-specific differences in transduction efficiency did not directly correlate with viral binding to muscle cells but rather appeared to involve endocytic or intracellular barriers to infection. Furthermore, application of this pseudotyped virus in a mouse model of Duchenne's muscular dystrophy also demonstrated significantly improved transduction efficiency. These findings should have a significant impact on improving rAAV-mediated gene therapy in muscle.

scholarly journals Machine Learning Models for Accurate Prioritization of Variants of Uncertain Significance

2020 ◽  
Author(s):  
Daniel Mahecha ◽  
Haydemar Nuñez ◽  
Maria Lattig ◽  
Jorge Duitama
Keyword(s):  
Machine Learning ◽  
Genetic Diagnosis ◽  
Software Tool ◽  
Support Vector ◽  
Clinical Settings ◽  
Loss Of Function ◽  
Variants Of Uncertain Significance ◽  
Sequencing Technologies ◽  
Uncertain Significance ◽  
New Generation

The growing use of new generation sequencing technologies on genetic diagnosis has produced an exponential increase in the number of Variants of Uncertain Significance (VUS). In this manuscript we compare three machine learning methods to classify VUS as Pathogenic or No pathogenic, implementing a Random Forest (RF), a Support Vector Machine (SVM), and a Multilayer Perceptron (MLP). To train the models, we extracted 82,463 high quality variants from ClinVar, using 9 conservation scores, the loss of function tool and allele frequencies. For the RF and SVM models, hyperparameters were tuned using cross validation with a grid search. The three models were tested on a set of 5,537 variants that had been classified as VUS any time along the last three years but had been reclassified in august 2020. The three models yielded superior accuracy on this set compared to the benchmarked tools. The RF based model yielded the best performance across different variant types and was used to create VusPrize, an open source software tool for prioritization of variants of uncertain significance. We believe that our model can improve the process of genetic diagnosis on research and clinical settings.

Targeted Modifications in Adeno-Associated Virus Serotype (AAV)- 8 Capsid Improves Its Hepatic Gene Transfer Efficiency in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2045-2045
Author(s):  
Dwaipayan Sen ◽  
Nishanth Gabriel ◽  
Sathish Kumar Yesupatham ◽  
Rekha Samuel ◽  
Rupali A Gadkari ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Conflicts Of Interest ◽  
Innate Immune ◽  
Hemophilia B ◽  
Transduction Efficiency ◽  
Egfp Expression ◽  
Adeno Associated Virus ◽  
Virus Serotype ◽  
Antibody Levels

Abstract Abstract 2045 Recombinant adeno-associated virus vectors based on serotype (AAV)-8 have shown significant promise for liver directed gene therapy of hemophilia B. However, in a recent clinical trial, two patients who received highest dose (2×1012 vg/kg) of the self-complementary (sc)AAV8 vector developed capsid specific T cells that required glucocorticoid therapy to attenuate this response [Nathwani et al, New Eng J Med, 2011]. Thus, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well. It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal degradation machinery, we modified specific serine/threonine kinase or ubiquitination targets on AAV8 capsid to improve its transduction efficiency. To test this, point mutations at specific serine (S), threonine (T) or lysine (K) residues were generated on AAV8 capsid. scAAV8-EGFP vectors containing the wild-type (WT) and each one of the 5 S/T/K-mutant capsids were evaluated for their liver transduction efficiency at a dose of 5 × 1010 vgs/ animal in C57BL/6 mice in vivo. Two of the AAV8-S>A mutants (S279A and S501A) and a K137R mutant vector, demonstrated significantly higher EGFP expression (3.6 to 12.5 fold) in the liver compared to animals that received WT-AAV8 vectors alone (Figure 1). The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response [interleukin (IL)-6, IL-12, tumor necrosis factor α, Kupffer cells (KC) and innate immune responsive toll like receptors (TLR)-9] with a concomitant 2-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies also revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (22 fold), lungs (9.7 fold) and muscle (8.4 fold) tissue when compared to WT-AAV8 vectors. Further on-going studies with the optimal mutant scAAV8 vector expressing human coagulation factor IX in murine models of hemophilia B, will demonstrate the feasibility of the use of these novel vectors for potential gene therapy of hemophilia B. Figure 1: Efficacy of novel AAV8 S>A and K>R vectors (A) EGFP expression in hepatocytes 4 weeks post administration of AAV8 vectors in C57BL/6 mice, (B) Neutralization antibody levels against AAV8 vectors (C) Ubiquitination levels of K137R-AAV8 compared to the WT-AAV8 vector. Figure 1:. Efficacy of novel AAV8 S>A and K>R vectors (A) EGFP expression in hepatocytes 4 weeks post administration of AAV8 vectors in C57BL/6 mice, (B) Neutralization antibody levels against AAV8 vectors (C) Ubiquitination levels of K137R-AAV8 compared to the WT-AAV8 vector. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immune responses to adeno-associated virus type 2 encoding channelrhodopsin-2 in a genetically blind rat model for gene therapy

Gene Therapy ◽  
2010 ◽  
Vol 18 (3) ◽  
pp. 266-274 ◽  
Author(s):  
E Sugano ◽  
H Isago ◽  
Z Wang ◽  
N Murayama ◽  
M Tamai ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Responses ◽  
Virus Type ◽  
Rat Model ◽  
Adeno Associated Virus

scholarly journals Co-Prevalence of Pre-Existing Immunity to Different Serotypes of Adeno-Associated Virus (AAV) in Adults with Hemophilia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3349-3349
Author(s):  
Kavitha Rajavel ◽  
Mila Ayash-Rashkovsky ◽  
Ying Tang ◽  
Bagirath Gangadharan ◽  
Maurus de la Rosa ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
T Cell ◽  
Immune Responses ◽  
Peripheral Blood ◽  
Cellular Response ◽  
Employment Equity ◽  
Adeno Associated Virus ◽  
Peptide Antigens ◽  
Equity Ownership

Background: Recombinant and synthetic adeno-associated virus (AAV) vectors are in development for gene transfer in patients with hemophilia A (HA) or hemophilia B (HB). These include liver-directed recombinant AAV8 vectors BAX 888/SHP654/TAK-754 factor VIII (FVIII) gene therapy (GT) for severe HA, and SHP648/TAK-748 factor IX (FIX) GT for HB (Baxalta US Inc., a Takeda company, Lexington, MA, USA). However, environmental exposure to wild-type AAVs can result in individuals developing antibodies and cell-mediated immune responses to the naturally occurring AAV. While natural exposure to AAV does not result in any known disease in humans, presence of preexisting immunity can block delivery and prevent sustained expression of the transgene by an AAV-based vector in a gene therapy setting. Of the AAV serotypes, AAV2 is the most frequently encountered natural human infection and AAV5 and AAV8 have been the most commonly used vectors for hemophilia GT. Therefore, it is important to assess the prevalence and co-prevalence of antibody and T cell-mediated responses against each of these AAV serotypes and to better characterize the association between humoral and cellular immunity in people with hemophilia. Aims: To determine the prevalence of preexisting antibody-mediated immunity against AAV2, AAV5 and AAV8 and the association between AAV8-specific humoral and cell-mediated responses in adult patients with HA and HB in an international prospective, epidemiological study. Methods: This ongoing seroprevalence study involved adult male patients (18-75 years of age) with severe HA (<1% plasma FVIII activity) or severe/moderate HB (≤2% plasma FIX activity) recruited from hemophilia treatment centers in the United States and Europe (NCT03185897). Participants consented to collection of peripheral blood at either a single or multiple annual outpatient study visits, in order to explore fluctuations of the immune response over time. Local ethics committee approval was obtained. Titers for anti-AAV2, anti-AAV5 and anti-AAV8 neutralizing antibodies (NAbs) were determined using a cell-based transduction inhibition assay, with seropositivity defined as a titer ≥1:5. Titers for anti-AAV2, anti-AAV5 and anti-AAV8 binding antibodies (BAbs) were quantitated by indirect enzyme-linked immunosorbent assay (ELISA), with seropositivity defined as a titer ≥1:80. Cell-mediated immune responses to AAV8 peptide antigens were measured in peripheral blood mononuclear cells using an interferon-γ enzyme-linked immunospot (ELISpot) assay. Samples with a signal ≥3 times background and >60 spots per million cells were defined as positive. Results: Here we present data from patients who completed at least a single visit at the time of the interim, one year data cut (November 9, 2018). Of 242 patients enrolled (mean ± SD age: 35.3 ± 11.4 years), 194 patients had HA and 48 patients had HB. The overall co-prevalence of NAbs and BAbs to AAV2, AAV5 and AAV8 was 39.7% (HA: 38.1%, 72/189; HB: 45.8%, 22/48) and 16.1% (HA: 16.5%, 31/188; HB: 14.6%, 7/48) respectively, with further details shown in Table 1. Overall, 38.3% of patients (82/214) exhibited a T cell-mediated immune response to AAV8 peptide antigens (HA: 35.9%, 61/170; HB: 47.7%, 21/44). Among patients with AAV-8-specific NAbs, 37.9% (39/103) demonstrated positive AAV8-specific ELISPOT results. (HA: 35.7%, 30/84; HB: 47.4%, 9/19). Conclusion: The findings from this ongoing study demonstrate that approximately 50% of patients with hemophilia have preexistent NAb responses to AAV2, AAV5 or AAV8 with 40% demonstrating co-prevalence to all 3 evaluated AAV serotypes. Similar percentages of patients exhibited a positive cellular response to AAV8 antigens. Further, patients with HB demonstrated a slightly higher co-prevalence and a higher cellular response than patients with HA. In the combined HA and HB cohorts, co-prevalence was almost 40% for AAV8-specific humoral and T-cell mediated immunity. These data will add to our appreciation of preexisting AAV immunity that prevent patient participation in gene therapy trials. Disclosures Rajavel: Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Ayash-Rashkovsky:Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Tang:Baxalta US Inc., a Takeda company: Employment, Equity Ownership. Gangadharan:Baxalta Innovations GmbH, a Takeda company: Employment. de la Rosa:Baxalta Innovations GmbH, a Takeda company: Employment. Ewenstein:Baxalta US Inc., a Takeda company: Employment, Equity Ownership, Other: a Takeda stock owner.

Stability of Recombinant Mosaic Adeno-Associated Virus Vector rAAV/DJ/CAG at Different Temperature Conditions

2021 ◽  
Vol 17 (11) ◽  
pp. 2114-2124
Author(s):  
Alicja Bie´nkowska-Tokarczyk ◽  
Maciej Małecki
Keyword(s):  
Gene Therapy ◽  
Uv Light ◽  
Temperature Fluctuations ◽  
Effect Of Temperature ◽  
Transduction Efficiency ◽  
Microscopy Imaging ◽  
Adeno Associated Virus ◽  
Targeted Gene Delivery ◽  
Gene Therapy Vectors ◽  
The Stability

The nanometer size and biological characteristics of recombinant adeno-associated virus vectors (rAAV) make them particularly useful as gene therapy vectors and they have been successfully used in this role. Our latest research revealed that the rAAV/DJ/CAG mosaic vector offers highly efficient targeted gene delivery to melanoma cells metastasized to the lungs and that the transduction is temperature dependent. In order to further explore the ability of the rAAV/DJ/CAG vector to deliver highly selective transduction, this study was designed to identify the transduction stability of rAAV/DJ/CAG under various conditions. The temperatures used in this study ranged from −196 ° (liquid nitrogen) to 90 °, and the effect of temperature fluctuations (freeze-thaw, cooling-heating cycles) was also studied. This research also investigated the effects of UV radiation (ultraviolet) on the rAAV/DJ/CAG activity. Changes in the transduction efficiency were assessed via fluorescence microscopy imaging and the qPCR method. Under the test conditions, the transduction efficiency was reduced by approx. 35%, on average. High temperatures (70 °/90 °) and UV light proved to have the most detrimental impact. Changes in the stability of the rAAV/DJ/CAG structure are manifested by variations in the number of genome copies (gc) and GFP+ cells. Temperature fluctuations resulted in differences in the number of gc while maintaining a similar number of GFP+ cells, which may indicate specific changes in the rAAV/DJ/CAG structure, triggering disorders or degradation in the vector entry. This study provides interesting insights into rAAV/DJ/CAG, and the implications of these findings provide a basis for developing new protocols in cancer gene therapy.

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