Adeno-Associated Virus Vector-Mediated Expression of Antirespiratory Syncytial Virus Antibody Prevents Infection in Mouse Airways.

Infants and older adults are especially vulnerable to infection by respiratory syncytial virus (RSV), which can cause significant illness and irreparable damage to the lower respiratory tract and for which an effective vaccine is not readily available. Palivizumab, a recombinant monoclonal antibody (mAb), is an approved therapeutic for RSV infection for use in high-risk infants only. Due to several logistical issues, including cost of goods and scale-up limitations, palivizumab is not approved for other populations that are vulnerable to severe RSV infections, such as older adults. In this study, we demonstrate that intranasal delivery of adeno-associated virus serotype 9 (AAV9) vector expressing palivizumab or motavizumab, a second-generation version of palivizumab, significantly reduced the viral load in the lungs of the BALB/c mouse model of RSV infection. Notably, we demonstrate that AAV9 vector-mediated prophylaxis against RSV was effective despite the presence of serum-circulating neutralizing AAV9 antibodies. These findings substantiate the feasibility of repeatedly administering AAV9 vector to the airway for seasonal prophylaxis against RSV, thereby expanding the application of vectored delivery of mAbs as an effective prophylaxis strategy against various airborne viruses.

Developing a second-generation clinical candidate AAV vector for gene therapy of familial hypercholesterolemia.

Gene therapy for hypercholesterolemia offers the potential to sustainably ameliorate disease for life with a single dose. In this study, we demonstrate the combinatorial effects of codon and vector optimization, which significantly improve the efficacy of an adeno-associated virus (AAV) vector in the low-density lipoprotein receptor (LDLR)-deficient mouse model (Ldlr -/-, Apobec1 -/- double knockout [DKO]). This study investigated vector efficacy following the combination of intervening sequence 2 (IVS2) of the human beta-globin gene and codon optimization with the previously developed gain-of-function, human LDLR triple-mutant variant (hLDLR-L318D/K809R/C818A) in the treatment of homozygous familial hypercholesterolemia (HoFH). Vector doses as low as 3 × 1011 genome copies (GC)/kg achieved a robust reduction of serum low-density lipoprotein cholesterol (LDL-C) by 98% in male LDLR-deficient mice. Less efficient LDL-C reduction was observed in female mice, which was attributable to lower gene transfer efficiency in liver. We also observed persistent and stable transgene expression for 120 days, with LDL-C levels being undetectable in male DKO mice treated with the second-generation vector. In conclusion, codon and vector optimization enhanced transgene expression and reduced serum LDL-C levels effectively at a lower dose in LDLR-deficient mice. The second-generation clinical candidate vector we have developed has the potential to achieve therapeutic effects in HoFH patients.

Isolating Human Monoclonal Antibodies Against Adeno-Associated Virus From Donors With Pre-existing Immunity.

With the advent of single B-cell cloning technology, we can isolate antibodies against virtually any antigen to study the interaction of a given pathogen with the immune system and develop novel therapeutic strategies. Antibodies directed against the capsid of adeno-associated viruses (AAV) are a significant obstacle to effectively leveraging AAV as a gene-delivery vector in seropositive individuals. In order to design next-generation vectors that can evade neutralization by these antibodies, studies have mapped the epitopes of mouse monoclonal antibodies generated by immunization with AAV. Although these studies provide critical information regarding capsid immunogenicity, they cannot address (1) differences in the antibody repertoire generated in humans following AAV natural infection; or (2) how reactions can vary when generated in response to vector administration. Here, we isolated and evaluated a panel of novel, fully human anti-AAV antibodies by cloning single memory B cells from a seropositive normal donor. We have validated the utility of this approach to study AAV immunology. Our goal is to leverage this knowledge to design novel AAV variants that can effectively transduce target tissues in individuals with AAV-neutralizing antibodies.

Adeno-associated virus-mediated expression of human butyrylcholinesterase to treat organophosphate poisoning.

Rare diseases defined by genetic mutations are classic targets for gene therapy. More recently, researchers expanded the use of gene therapy in non-clinical studies to infectious diseases through the delivery of vectorized antibodies to well-defined antigens. Here, we further extend the utility of gene therapy beyond the "accepted" indications to include organophosphate poisoning. There are no approved preventives for the multi-organ damage resulting from acute or chronic exposure to organophosphates. We show that a single intramuscular injection of adeno-associated virus vector produces peak expression (~0.5 mg/ml) of active human butyrylcholinesterase (hBChE) in mice serum within 3-4 weeks post-treatment. This expression is sustained for up to 140 days post-injection with no silencing. Sustained expression of hBChE provided dose-dependent protection against VX in male and female mice despite detectable antibodies to hBChE in some mice, thereby demonstrating that expression of hBChE in vivo in mouse muscle is an effective prophylactic against organophosphate poisoning.

Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin.

Broadly neutralizing antibodies against highly variable pathogens have stimulated the design of vaccines and therapeutics. We report the use of diverse camelid single-domain antibodies to influenza virus hemagglutinin to generate multidomain antibodies with impressive breadth and potency. Multidomain antibody MD3606 protects mice against influenza A and B infection when administered intravenously or expressed locally from a recombinant adeno-associated virus vector. Crystal and single-particle electron microscopy structures of these antibodies with hemagglutinins from influenza A and B viruses reveal binding to highly conserved epitopes. Collectively, our findings demonstrate that multidomain antibodies targeting multiple epitopes exhibit enhanced virus cross-reactivity and potency. In combination with adeno-associated virus-mediated gene delivery, they may provide an effective strategy to prevent infection with influenza virus and other highly variable pathogens.

Intrathecal Viral Vector Delivery of Trastuzumab Prevents or Inhibits Tumor Growth of Human HER2-Positive Xenografts in Mice.

Breast cancer brain metastases are a deadly sequela of primary breast tumors that overexpress human epidermal growth factor receptor 2 (HER2); median survival for patients with these tumors is 10 to 13 months from the time of diagnosis. Current treatments for HER2-positive breast cancer brain metastases are invasive, toxic, and largely ineffective. Here, we have developed an adeno-associated virus serotype 9 (AAV9) vector to express the anti-HER2 monoclonal antibody trastuzumab (Herceptin) in vivo A single prophylactic intrathecal administration of AAV9.trastuzumab vector in a novel orthotopic Rag1-/- murine xenograft model of HER2-positive breast cancer brain metastases significantly increased median survival, attenuated brain tumor growth, and preserved both the HER2 antigen specificity and the natural killer cell-associated mechanism of action of trastuzumab. When administered as a tumor treatment, AAV9.trastuzumab increased median survival. Dose-escalation studies revealed that higher doses of AAV9.trastuzumab resulted in smaller tumor volumes. Our results indicate that intrathecal AAV9.trastuzumab may provide significant antitumor activity in patients with HER2-positive breast cancer brain metastases.Significance: Intrathecal delivery of trastuzumab via adeno-associated virus has the potential to become a novel, integral part of adjuvant therapy for patients with HER2-positive breast cancer brain metastases. Cancer Res; 78(21); 6171-82. ©2018 AACR.

Intramuscular administration of AAV overcomes pre-existing neutralizing antibodies in rhesus macaques.

The seroprevalence of neutralizing antibodies (NAbs) to adeno-associated viral (AAV) vector capsids may preclude a percentage of the population from receiving gene therapy, particularly following systemic vector administration. We hypothesized that the use of intramuscular (IM) administration of AAV vectors might circumvent this issue. IM injections were used to administer AAV8 vectors expressing either secreted or non-secreted transgenes into mice and the influence of NAbs supplied by pre-administration of pooled human IgG on transgene expression was evaluated. We then studied the impact of naturally occurring pre-existing AAV8 NAbs on expression of a secreted transgene following IM vector delivery in rhesus macaques. Finally, we evaluated the ability to readminister AAV vectors via IM injections in rhesus macaques. In mice, the presence of AAV8 NAbs had no effect on gene expression in the injected skeletal muscle. However, liver transgene expression following hepatic distribution of the vector was ablated. In rhesus macaques, naturally occurring pre-existing AAV8 NAb titers of ⩽1:160 had no effect on expression levels of a secreted transgene after IM delivery of the vector. Additionally, readministration of AAV vectors was possible by IM injection into the previously injected muscle groups, with no effect on transgene expression by the original vector. Therefore, the presence of pre-existing NAbs in the human population should not preclude subjects from receiving gene therapy by IM administration of the vector so long as sufficient levels of secreted transgene expression can be produced without the involvement of liver.

Intramuscular injection of AAV8 in mice and macaques is associated with substantial hepatic targeting and transgene expression.

Intramuscular (IM) administration of adeno-associated viral (AAV) vectors has entered the early stages of clinical development with some success, including the first approved gene therapy product in the West called Glybera. In preparation for broader clinical development of IM AAV vector gene therapy, we conducted detailed pre-clinical studies in mice and macaques evaluating aspects of delivery that could affect performance. We found that following IM administration of AAV8 vectors in mice, a portion of the vector reached the liver and hepatic gene expression contributed significantly to total expression of secreted transgenes. The contribution from liver could be controlled by altering injection volume and by the use of traditional (promoter) and non-traditional (tissue-specific microRNA target sites) expression control elements. Hepatic distribution of vector following IM injection was also noted in rhesus macaques. These pre-clinical data on AAV delivery should inform safe and efficient development of future AAV products.

5-HT(1B) receptors play a prominent role in the proliferation of T-lymphocytes.

Serotonin plays a role in T cell activation, but there is no clear consensus of which of the 14 serotonergic receptors control this activations pathway. We have used a broad range of serotonergic receptor antagonists to define the functional involvement of these receptors governing the proliferation of primary T cells as well as in T cell lines. Our data shows that antagonism of the 5-HT(1B) receptor inhibits the proliferation of both human and murine primary helper T cells and of human helper T cell lines. As a whole, our data suggest that other serotonergic receptors may contribute to the proliferative signals, but the 5-HT(1B) receptor plays the most dominant role.