scholarly journals Immunoadsorption enables successful rAAV5-mediated repeated hepatic gene delivery in nonhuman primates

2019 ◽  
Vol 3 (17) ◽  
pp. 2632-2641 ◽  
Author(s):  
David Salas ◽  
Karin L. Kwikkers ◽  
Nerea Zabaleta ◽  
Andrea Bazo ◽  
Harald Petry ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Neutralizing Antibodies ◽  
Nonhuman Primates ◽  
Human Subjects ◽  
Factor Ix ◽  
Target Cells ◽  
Targeted Gene Delivery ◽  
Human Factor Ix ◽  
Relevant Animal Model ◽  
Liver Gene

Abstract Adeno-associated virus (AAV)–based liver gene therapy has been shown to be clinically successful. However, the presence of circulating neutralizing antibodies (NABs) against AAV vector capsids remains a major challenge as it may prevent successful transduction of the target cells. Therefore, there is a need to develop strategies that would enable AAV-mediated gene delivery to patients with preexisting anti-AAV NABs. In the current study, the feasibility of using an immunoadsorption (IA) procedure for repeated, liver-targeted gene delivery in nonhuman primates was explored. The animals were administered IV with recombinant AAV5 (rAAV5) carrying the reporter gene human secreted embryonic alkaline phosphatase (hSEAP). Seven weeks after the first rAAV treatment, all of the animals were readministered with rAAV5 carrying the therapeutic hemophilia B gene human factor IX (hFIX). Half of the animals administered with rAAV5-hSEAP underwent IA prior to the second rAAV5 exposure. The transduction efficacies of rAAV5-hSEAP and rAAV5-hFIX were assessed by measuring the levels of hSEAP and hFIX proteins. Although no hFIX was detected after rAAV5-hFIX readministration without prior IA, all animals submitted to IA showed therapeutic levels of hFIX expression, and a threshold of anti-AAV5 NAB levels compatible with successful readministration was demonstrated. In summary, our data demonstrate that the use of a clinically applicable IA procedure enables successful readministration of an rAAV5-based gene transfer in a clinically relevant animal model. Finally, the analysis of anti-AAV NAB levels in human subjects submitted to IA confirmed the safety and efficacy of the procedure to reduce anti-AAV NABs. Furthermore, clinical translation was assessed using an immunoglobulin G assay as surrogate.

scholarly journals Development of Monospecific Antibodies to Human Factor IX

1977 ◽  
Author(s):  
Cheryl Y. Tiarks ◽  
Chin-Hai Chang ◽  
Liberto Pechet
Keyword(s):  
Neutralizing Antibodies ◽  
Human Factor ◽  
Human Albumin ◽  
Factor Ix ◽  
Hemophilia B ◽  
Red Cross ◽  
Factor X ◽  
Normal Plasma ◽  
Human Factor Ix ◽  
Monospecific Antibodies

The purpose of this research was to develop neutralizing and precipitating antibodies to factor IX. Human factor IX, purified by the method of Rosenberg et.al. (J. Biol. Chem. 250:8883, 1975), was electrophoresed on acrylamide gel. Two major bands migrating adjacently were eluted. They contained factor IX activity only. The eluates and their homogenized gel segments 7 and 8 were injected separately into two rabbits, Rl and R2, respectively. On immunodiffusion the antiserum Rl showed one precipitating line with normal plasma. It neutralized human factor IX (20 Bethesda units) and also slightly neutralized factor X. It had no effect on factors II and VII. Following absorption of this antiserum with purified factor X it neutralized factor IX only. With continuous immunization, however, this antiserum revealed two new precipitating contaminants. The antiserum R2 neutralized only factor IX; it reached 220 Bethesda inhibitory units. On immunodiffusion it showed two precipitating lines, one of which disappeared after absorption with human albumin. On immunodiffusion and Laurell immunoelectrophoresis, the albumin-absorbed R2 antiserum showed one precipitin line of identity, or one rocket, with normal plasma, a Red Cross factor IX preparation (rich in factors IX, II and X), the original eluates 7 and 8, and a Hemophilia-B antigen-positive plasma. No line or rocket developed with normal plasma absorbed with aluminum hydroxide or with antigen-negative Hemophilia-B plasma. We conclude that the antisera Rl and R2 contain factor IX neutralizing antibodies and that albumin-absorbed R2 has monospecific precipitating antibodies to human non-activated factor IX.

scholarly journals Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX

Gene Therapy ◽  
2005 ◽  
Vol 13 (2) ◽  
pp. 117-126 ◽  
Author(s):  
B W Bigger ◽  
E K Siapati ◽  
A Mistry ◽  
S N Waddington ◽  
M S Nivsarkar ◽  
...  
Keyword(s):  
Stem Cell ◽  
Gene Delivery ◽  
Mouse Model ◽  
Human Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Human Factor Ix ◽  
Cell Gene ◽  
Stem Cell Gene

Rabbit Anti-Thymocyte Globulin (rATG) Administrated Concomitantly with Liver Delivery of AAV2-hFIX Can Promote Inhibitor Formation In Rhesus Macaques.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3765-3765 ◽  
Author(s):  
Jonathan D. Finn ◽  
Patricia Favaro ◽  
J. Fraser Wright ◽  
Federico Mingozzi ◽  
Katherine A. High ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Transfer ◽  
Neutralizing Antibodies ◽  
Rhesus Macaques ◽  
Rescue Therapy ◽  
Factor Ix ◽  
Large Animal ◽  
Solid Organ ◽  
Large Animal Models ◽  
Human Factor Ix

Abstract Abstract 3765 Adeno-associated viral (AAV) vectors are one of the most extensively studied vector platforms for gene therapy applications. Our group is currently developing AAV vectors for the therapeutic treatment of hemophilia B (HB) in humans. The first clinical trial using an AAV2 vector to express human Factor IX (hFIX) (AAV2-hFIX16) from the liver of HB patients revealed a cytotoxic T lymphocyte (CTL) response directed against AAV capsid that occurred 4–6 weeks following treatment that was associated with a decline in transgene expression. Thus, immunosuppressive (IS) therapies may be required during AAV2 vector administration at high doses to prevent or to halt the immune mediated destruction of transduced hepatocytes. Previous work in murine and non-human primate (NHP) models has shown that sustained AAV-mediated expression of transgenes can induce tolerance, and that this is in part, dependent on CD4+ CD25+ FoxP3+ regulatory T cells (Tregs). Here we investigate the safety of a Treg sparing anti-T cell IS regimen in the context of liver mediated AAV2 gene transfer. Rabbit anti-thymocyte globulin (rATG) is an immune suppressive drug that is used in solid organ transplant and autoimmune disease. rATG has been shown to dramatically deplete the majority of T-cells, however some studies have shown that rATG spares Tregs and can induce tolerance in human T cells. rATG was administered to rhesus macaques (along with an 8-week course of Mycophenolate Mofetil (MMF) and sirolimus) either at the time of AAV vector administration (AAV2-hFIX16), or 5 weeks post-vector administration (rescue therapy). The administration of ATG at week 5 had no detrimental effect on hFIX expression and was not associated with inhibitor formation (n=3) indicating that rATG might be safe to use as an IS ‘rescue' agent, after the detection of an ongoing immune response against transduced cells. Interestingly we observed that early administration of rATG prevented tolerance induction and resulted in inhibitor formation in 2 of 3 animals upon withdrawal of IS. The inhibitor formation was associated with transient elevations in circulating levels of IL-2, IL-4, IL-10 and IFN-g. These results are comparable to previous findings in NHP using an anti-CD25 IS regimen (Daclizumab) at the time of vector administration (Blood 2007, 110(7):2334-41). We conclude that the timing of IS regimens is critical, and that IS regimens that alter the numbers, frequency, and/or function of T-cells at the time of vector administration can result in neutralizing antibodies (inhibitors) to the transgene product (hFIX). These data suggest that there might be multiple mechanisms responsible for maintaining tolerance in this model, and that Tregs alone might not be sufficient. This study highlights the critical need for safety studies in large animal models of potential immune suppressive regimens in the context of gene transfer before translating to the clinic. Disclosures: High: Genzyme, Inc: Consultancy, Patents & Royalties; Third Rock Ventures: Consultancy; Novo-Nordisk: Consultancy; Shire, Inc.: Consultancy.

Ultrasound Enhances Gene Delivery of Human Factor IX Plasmid

2005 ◽  
Vol 16 (7) ◽  
pp. 893-905 ◽  
Author(s):  
Carol H. Miao ◽  
Andrew A. Brayman ◽  
Keith R. Loeb ◽  
Peiqing Ye ◽  
Ling Zhou ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Human Factor ◽  
Factor Ix ◽  
Human Factor Ix

scholarly journals Efficient and Highly Specific Gene Transfer Using Mutated Lentiviral Vectors Redirected with Bispecific Antibodies

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Christina L. Parker ◽  
Timothy M. Jacobs ◽  
Justin T. Huckaby ◽  
Dimple Harit ◽  
Samuel K. Lai
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Endosomal Escape ◽  
Bispecific Antibodies ◽  
Target Cells ◽  
Specific Gene ◽  
Targeted Gene Delivery

ABSTRACT Despite their exceptional potencies, the broad tropism of most commonly used lentivirus (LV) vectors limits their use for targeted gene delivery in vivo. We hypothesized that we could improve the specificity of LV targeting by coupling (i) reduction of their binding to off-target cells with (ii) redirection of the vectors with a bispecific antibody (bsAb) that binds both LV and receptors on target cells. As a proof of concept, we pseudotyped nonreplicating LV using a mutated Sindbis envelope (mSindbis) with ablated binding to native receptors, while retaining the capacity to facilitate efficient fusion and endosomal escape. We then evaluated the transduction potencies of the mSindbis LV for HER2-positive (HER2+) (SKBR3) breast and HER2-negative (HER2−) (A2780) cells when redirected with different bsAbs. mSindbis LV alone failed to induce appreciable green fluorescent protein (GFP) expression in either cell. When mixed with HER2-targeting bsAb, mSindbis LV was exceptionally potent, transducing 12% to 16% of the SKBR3 cells at a multiplicity of infection (MOI [ratio of viral genome copies to target cells]) of 3. Transduction was highly specific, resulting in ∼50-fold-greater selectivity toward SKBR3 cells versus A2780 cells. Redirecting mSindbis LV led to a 10-fold improvement in cell-specific targeting compared to redirecting wild-type Sindbis LV with the same bsAb, underscoring the importance of ablating native virus tropism in order to maximize targeting specificity. The redirection of mutated LV using bsAb represents a potent and highly versatile platform for targeted gene therapy. IMPORTANCE The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentivirus (LV) vectors are efficient gene delivery vehicles but offer little specificity. Here, we report an effective and versatile strategy to redirect LV to target cells using bispecific antibodies (bsAbs) that bind both cell receptors and LV envelope domains. Importantly, we ablated the native receptor binding of LV to minimize off-target transduction. Coupling bsAb specificity and ablated native LV tropism synergistically enhanced the selectivity of our targeted gene delivery system. The modular nature of our bsAb-based redirection enables facile targeting of the same LV to diverse tissues/cells. By abrogating the native broad tropism of LV, our bsAb-LV redirection strategy may enable lentivirus-based gene delivery in vivo, expanding the current use of LV beyond ex vivo applications.

In utero gene transfer of human factor IX to fetal mice can induce postnatal tolerance of the exogenous clotting factor

Blood ◽  
2003 ◽  
Vol 101 (4) ◽  
pp. 1359-1366 ◽  
Author(s):  
Simon N. Waddington ◽  
Suzanne M. K. Buckley ◽  
Megha Nivsarkar ◽  
Sarah Jezzard ◽  
Holm Schneider ◽  
...  
Keyword(s):  
Immune Tolerance ◽  
Human Factor ◽  
Adult Life ◽  
Factor Ix ◽  
Clotting Factor ◽  
Target Cells ◽  
Proliferating Cells ◽  
Disease Manifestation ◽  
Adult Mice ◽  
Human Factor Ix

The fundamental hypotheses behind fetal gene therapy are that it may be possible (1) to achieve immune tolerance of transgene product and, perhaps, vector; (2) to target cells and tissues that are inaccessible in adult life; (3) to transduce a high percentage of rapidly proliferating cells, and in particular stem cells, with relatively low absolute virus doses leading to clonal transgene amplification by integrating vectors; and (4) to prevent early disease manifestation of genetic diseases. This study provides evidence vindicating the first hypothesis; namely, that intravascular prenatal administration of an adenoviral vector carrying the human factor IX (hFIX) transgene can induce immune tolerance of the transgenic protein. Following repeated hFIX protein injection into adult mice, after prenatal vector injection, we found persistence of blood hFIX and absence of hFIX antibodies in 5 of 9 mice. Furthermore, there was substantial hFIX expression after each of 2 reinjections of vector without detection of hFIX antibodies. In contrast, all adult mice that had not been treated prenatally showed a rapid loss of the injected hFIX and the development of high hFIX antibody levels, both clear manifestations of a strong immune reaction.

AAV8-Factor IX Hepatic Gene Transfer with Transient Immunosuppression Is Safe but Is Abrogated by Low Titers of Neutralizing Antibody in Rhesus Macaques.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5532-5532
Author(s):  
Haiyan Jiang ◽  
Susannah Patarroyo-White ◽  
Tongyao Liu ◽  
Bin Yang ◽  
Dea Nagy ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Neutralizing Antibodies ◽  
Rhesus Macaques ◽  
Small Animal ◽  
Neutralizing Antibody ◽  
Cross Reactivity ◽  
Factor Ix ◽  
Post Surgery ◽  
Short Course ◽  
Human Factor Ix

Abstract AAV8 vectors have potential in human gene transfer due to improved transduction efficiencies as demonstrated in small animal models, and are also reported to show less cross-reactivity to AAV2 neutralizing antibodies prevalent in humans. To assess this hypothesis, the efficacy and safety of AAV8-human Factor IX (hFIX) was evaluated in rhesus macaques. At doses of 5–20x1012 vg/kg, AAV8-hFIX achieved therapeutic to supraphysiological levels of circulating human FIX via intra-hepatic artery delivery in AAV8-naïve macaques. However, transduction was abrogated in macaques with pre-existing AAV8 neutralizing antibodies at titers as low as 1:5. Intrahepatic delivery of AAV8-hFIX resulted in gene transfer primarily in liver, and was well distributed among individual hepatic lobes. Two macaques that experienced traumatic catheterization developed immediate and delayed (week 4–6) transient transaminitis post surgery. Nevertheless, a significant percentage of AAV8-hFIX transduced hepatocytes persisted despite the liver damage and partial loss of hFIX expression. Transduction was well tolerated in all other macaques. Transient immunosuppression with tacrolimus and mycophenolate mofetil did not impair AAV8-hFIX transduction. The results [1] support AAV8-mediated gene transfer in humans but indicate that both the efficacy achieved and the resistance to pre-existing neutralizing antibodies of AAV8-hFIX are unlikely to be significantly enhanced over AAV2 vector, [2] suggest that a peri-operative insult and likely triggering of innate immunity may result in a later inflammatory response that results in a decrease in transgene protein production, and [3] provide evidence that a short course of immunosuppression does not alter liver transduction by AAV vectors.

scholarly journals CRISPR/Cas9-mediated in vivo gene targeting corrects hemostasis in newborn and adult factor IX–knockout mice

Blood ◽  
2019 ◽  
Vol 133 (26) ◽  
pp. 2745-2752 ◽  
Author(s):  
Lili Wang ◽  
Yang Yang ◽  
Camilo Ayala Breton ◽  
John White ◽  
Jia Zhang ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Partial Hepatectomy ◽  
Neutralizing Antibodies ◽  
Genetic Diseases ◽  
Factor Ix ◽  
Therapeutic Effects ◽  
Vector Genome ◽  
Human Factor Ix

Abstract Many genetic diseases, including hemophilia, require long-term therapeutic effects. Despite the initial success of liver-directed adeno-associated virus (AAV) gene therapy for hemophilia in clinical trials, long-term sustained therapeutic effects have yet to be seen. One explanation for the gradual decline of efficacy over time is that the nonintegrating AAV vector genome could be lost during cell division during hepatocyte turnover, albeit at a slow pace in adults. Readministering the same vector is challenging as a result of the AAV-neutralizing antibodies elicited by the initial treatment. Here, we investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated homology-directed gene targeting for sustained treatment of hemophilia B. We developed a donor vector containing a promoterless partial human factor IX (FIX) complementary DNA carrying the hyperactive FIX Padua mutation. A single injection of dual AAV vectors in newborn and adult FIX-knockout (FIX-KO) mice led to stable expression of FIX at or above the normal levels for 8 months. Eight weeks after the vector treatment, we subjected a subgroup of newborn and adult treated FIX-KO mice to a two-thirds partial hepatectomy; all of these animals survived the procedure without any complications or interventions. FIX levels persisted at similar levels for 24 weeks after partial hepatectomy, indicating stable genomic targeting. Our results lend support for the use of a CRISPR/Cas9 approach to achieve lifelong expression of therapeutic proteins.

Sign in / Sign up

Export Citation Format

Share Document