Structure comparison of the chimeric AAV2.7m8 vector with parental AAV2.

The AAV2.7m8 vector is an engineered capsid with a 10-amino acid insertion in adeno-associated virus (AAV) surface variable region VIII (VR-VIII) resulting in the alteration of an antigenic region of AAV2 and the ability to efficiently transduce retina cells following intravitreal administration. Directed evolution and in vivo screening in the mouse retina isolated this vector. In the present study, we sought to identify the structural differences between a recombinant AAV2.7m8 (rAAV2.7m8) vector packaging a GFP genome and its parental serotype, AAV2, by cryo-electron microscopy (cryo-EM) and image reconstruction. The structures of rAAV2.7m8 and AAV2 were determined to 2.91 and 3.02 Å resolution, respectively. The rAAV2.7m8 amino acid side-chains for residues 219-745 (the last C-terminal residue) were interpretable in the density map with the exception of the 10 inserted amino acids. While observable in a low sigma threshold density, side-chains were only resolved at the base of the insertion, likely due to flexibility at the top of the loop. A comparison to parental AAV2 (ordered from residues 217-735) showed the structures to be similar, except at some side-chains that had different orientations and, in VR-VIII containing the 10 amino acid insertion. VR-VIII is part of an AAV2 antigenic epitope, and the difference is consistent with rAAV2.7m8's escape from a known AAV2 monoclonal antibody, C37-B. The observations provide valuable insight into the configuration of inserted surface peptides on the AAV capsid and structural differences to be leveraged for future AAV vector rational design, especially for retargeted tropism and antibody escape.

Preclinical Evaluation of ADVM-022, a Novel Gene Therapy Approach to Treating Wet Age-Related Macular Degeneration.

Inhibition of vascular endothelial growth factor, a key contributor to the choroidal neovascularization associated with wet age-related macular degeneration, is the mode of action of several approved therapies, including aflibercept, which requires frequent intravitreal injections to provide clinical benefit. Lack of compliance with the dosing schedule may result in recurrence of active wet macular degeneration, leading to irreversible vision impairment. Gene therapy providing sustained anti-vascular endothelial growth factor levels in the retina following a single injection could drastically reduce the treatment burden and improve visual outcomes. ADVM-022, an adeno-associated virus vector encoding aflibercept, is optimized for intravitreal delivery and strong protein expression. Here, we report the long-term expression and efficacy of ADVM-022-derived aflibercept in a laser-induced choroidal neovascularization model in non-human primates. Intravitreal administration of ADVM-022 was well tolerated and resulted in sustained aflibercept levels. In addition, ADVM-022 administration 13 months before lasering prevented the occurrence of clinically relevant choroidal neovascularization lesions, similar to animals that received a bolus of intravitreal aflibercept (standard of care) at the time of lesioning. These results demonstrate that a single intravitreal administration of ADVM-022 may provide a safe and effective long-term treatment option for wet macular degeneration and may ultimately improve patients' visual outcomes.

Acetylcholine receptor based chemogenetics engineered for neuronal inhibition and seizure control assessed in mice.

Epilepsy is a prevalent disorder involving neuronal network hyperexcitability, yet existing therapeutic strategies often fail to provide optimal patient outcomes. Chemogenetic approaches, where exogenous receptors are expressed in defined brain areas and specifically activated by selective agonists, are appealing methods to constrain overactive neuronal activity. We developed BARNI (Bradanicline- and Acetylcholine-activated Receptor for Neuronal Inhibition), an engineered channel comprised of the α7 nicotinic acetylcholine receptor ligand-binding domain coupled to an α1 glycine receptor anion pore domain. Here we demonstrate that BARNI activation by the clinical stage α7 nicotinic acetylcholine receptor-selective agonist bradanicline effectively suppressed targeted neuronal activity, and controlled both acute and chronic seizures in male mice. Our results provide evidence for the use of an inhibitory acetylcholine-based engineered channel activatable by both exogenous and endogenous agonists as a potential therapeutic approach to treating epilepsy.

Matrix metalloproteinase-3 (MMP-3)-mediated gene therapy for glaucoma.

Approximately 80 million people globally are affected by glaucoma, with a projected increase to over 110 million by 2040. Substantial issues surrounding patient compliance remain with topical eye drops, and up to 10% of patients become treatment resistant, putting them at risk of permanent vision loss. The major risk factor for glaucoma is elevated intraocular pressure, which is regulated by the balance between the secretion of aqueous humor and the resistance to its flow across the conventional outflow pathway. Here, we show that adeno-associated virus 9 (AAV9)-mediated expression of matrix metalloproteinase-3 (MMP-3) can increase outflow in two murine models of glaucoma and in nonhuman primates. We show that long-term AAV9 transduction of the corneal endothelium in the nonhuman primate is safe and well tolerated. Last, MMP-3 increases outflow in donor human eyes. Collectively, our data suggest that glaucoma can be readily treated with gene therapy-based methods, paving the way for deployment in clinical trials.

Site-specific recombinase strategy to create induced pluripotent stem cells efficiently with plasmid DNA.

Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. iPSCs are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications. Many alternative strategies to make iPSCs have been developed, but the nonintegrating strategies tend to be inefficient, while the integrating strategies involve random integration. Here, we report a facile strategy to create murine iPSCs that uses plasmid DNA and single transfection with sequence-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette into the genome, producing iPSCs. Cre recombinase was then used for excision of the reprogramming genes. The iPSCs were demonstrated to be pluripotent by in vitro and in vivo criteria, both before and after excision of the reprogramming cassette. This strategy is comparable with retroviral approaches in efficiency, but is nonhazardous for the user, simple to perform, and results in nonrandom integration of a reprogramming cassette that can be readily deleted. We demonstrated the efficiency of this reprogramming and excision strategy in two accessible cell types, fibroblasts and adipose stem cells. This simple strategy produces pluripotent stem cells that have the potential to be used in a clinical setting.

Mutational derivatives of PhiC31 integrase with increased efficiency and specificity.

phiC31 integrase is a sequence-specific phage recombinase that can recombine two short DNA sequences called attB and attP. The enzyme can also promote genomic integration of plasmids carrying attB into native mammalian sequences having partial identity to attP. To increase the efficiency of integration, we mutated the phiC31 integrase gene and screened the mutants in human cells in an assay for higher recombination frequency between attB and attP. We report in this article the isolation of a mutant, P2 that has twice the chromosomal integration frequency of wild-type phiC31 integrase, at both a preintegrated chromosomal attP site and at endogenous pseudo attP sequences in cultured human cells. In mouse liver, P2-mediated integration provided therapeutic long-term levels of human factor IX that were double those generated by wild-type phiC31 integrase. We also describe an additional mutant, P3 that combines the mutations of P2 with further changes and possesses an elevated specificity for integration at a chromosomally placed attP site in human cells. Forty-four percent of colonies carrying integration events mediated by P3 have integration at the placed attP site. These mutant integrases are useful for gene therapy and genome modification, and they demonstrate the feasibility of engineering phiC31 integrase toward more desirable properties.

Systemic AAV9.LAMP2B injection reverses metabolic and physiologic multiorgan dysfunction in a murine model of Danon disease.

Danon disease (DD) is a rare X-linked autophagic vacuolar myopathy associated with multiorgan dysfunction, including the heart, skeletal muscle, and liver. There are no specific treatments, and most male patients die from advanced heart failure during the second or third decade of life. DD is caused by mutations in the lysosomal-associated membrane protein 2 (LAMP2) gene, a key mediator of autophagy. LAMP2 has three isoforms: LAMP2A, LAMP2B, and LAMP2C. LAMP2B is the predominant isoform expressed in cardiomyocytes. This study evaluates the efficacy of human LAMP2B gene transfer using a recombinant adeno-associated virus 9 carrying human LAMP2B (AAV9.LAMP2B) in a Lamp2 knockout (KO) mouse, a DD model. AAV9.LAMP2B was intravenously injected into 2- and 6-month-old Lamp2 KO male mice to assess efficacy in adolescent and adult phenotypes. Lamp2 KO mice receiving AAV9.LAMP2B demonstrated dose-dependent restoration of human LAMP2B protein in the heart, liver, and skeletal muscle tissue. Impaired autophagic flux, evidenced by increased LC3-II, was abrogated by LAMP2B gene transfer in all tissues in both cohorts. Cardiac function was also improved, and transaminases were reduced in AAV9.LAMP2B-treated KO mice, indicating favorable effects on the heart and liver. Survival was also higher in the older cohort receiving high vector doses. No anti-LAMP2 antibodies were detected in mice that received AAV9.LAMP2B. In summary, LAMP2B gene transfer improves metabolic and physiologic function in a DD murine model, suggesting that a similar therapeutic approach may be effective for treating patients with this highly morbid disease.

Long-term transgene expression in mouse neural progenitor cells modified with phiC31 integrase.

Stem cells can potentially be utilized in combined gene/cell therapies for neural diseases. We examined the ability of the non-viral phiC31 integrase system to promote stable transgene expression in mouse neural progenitor cells (mNPCs). phiC31 integrase catalyzes the sequence-specific integration of attB-containing plasmids into pseudo attP sites in mammalian genomes, to produce long-term transgene expression. We achieved gene transfer by co-nucleofection of a plasmid carrying the luciferase marker gene and an attB site and a plasmid expressing integrase in mNPCs that had been generated in a neurosphere preparation. Luciferase expression was quantified in live cells for 8 weeks, revealing persistence of gene expression. Sequence-specific integration at a preferred pseudo attP site in the mouse genome was detected by using PCR. Furthermore, sustained transgene expression was demonstrated in genetically modified NPCs that were cultured in conditions that promoted either growth or differentiation into neurons and astrocytes. Our results demonstrate that the phiC31 integrase system produces stable transgene expression in adult mNPCs and their progeny and may be useful in strategies for combating neurodegenerative disorders.

Site-specific chromosomal integration mediated by phiC31 integrase.

phiC31 integrase is a site-specific recombinase from a bacteriophage that has become a useful tool in mammalian cells. The enzyme normally performs precise, unidirectional recombination between two attachment or att sites called attB and attP. We have shown that an attP site preintegrated into a mammalian chromosome can serve as a target for integration of an introduced plasmid carrying an attB site. Recombination leads to precise integration of the plasmid into the chromosome at the attP site. This reaction is useful for placing introduced genes into the same chromosomal environment, in order to minimize position effects associated with random integration. Because phiC31 integrase can also mediate integration at endogenous sequences that resemble attP, called pseudo attP sites, a selection system is used that yields integration only at the desired preintegrated attP site. This chapter provides a protocol that features a simple antibiotic selection to isolate cell lines in which the introduced plasmid has integrated at the desired attP site. A polymerase chain reaction assay is also presented to verify correct chromosomal placement of the introduced plasmid. This integration system based on phiC31 integrase supplies a simple method to obtain repeated integration at the same chromosomal site in mammalian cells.

Hyperactive Himar1 transposase mediates transposition in cell culture and enhances gene expression in vivo.

The use of nonviral delivery systems results in transient gene expression, in part because of the low efficiency of DNA integration. Previously, vectors based on transposon systems such as Sleeping Beauty have been shown to be able to increase stable transfection efficiencies in cell culture and in animal models. Himar1, a reconstructed active transposon belonging to the Tc1/mariner superfamily, also has been used as a vector for stable gene delivery, but the rate of transposition after transfection is low. In this paper, we evaluate the potential of the hyperactive Himar1 transposase C9, in combination with the Himar1 inverted repeat transposon, as a gene delivery vector. The C9 transposase is a hyperactive mutant of Himar1 with two amino acid substitutions, Q131R and E137K, that result in an increase in activity relative to wild type. Here we demonstrate that cotransfection of the C9 transposase with a Himar1-based vector increases the frequency of stable gene expression in human cells in a transposase concentration-dependent manner. In addition, we establish that C9 transposase mediates integration of the transgene in mammalian cells at a frequency similar to that of Sleeping Beauty under some of the conditions tested. Last, we show significantly higher levels of reporter gene expression in vivo in mouse liver and in synovium of rabbit knee joints after injection of the transposon plasmid expressing the transgene and the C9 transposase. These data suggest that vectors based on the Himar1 transposable element, in conjunction with the hyperactive mutant transposase C9, may be suitable vectors for gene therapy applications.

Long-term increase in mVEGF164 in mouse hindlimb muscle mediated by phage phiC31 integrase after nonviral DNA delivery.

Peripheral vascular disease (PVD), characterized by insufficient blood supply to extremities, can be a devastating illness. Although many gene therapy strategies for PVD using vascular endothelial growth factor (VEGF) have resulted in increased blood vessel formation, the vessels are often impermanent and regress after therapy, probably because of the short-lived VEGF expression mediated by gene therapy vectors (14 days or less). phiC31 integrase is a recombinase originally isolated from a bacteriophage of Streptomyces. This integrase performs efficient chromosomal integration of plasmid DNA into mammalian genomes that results in long-term transgene expression. In this study, gene transfer was achieved by intramuscular injection of VEGF and integrase plasmid DNAs into the tibialis anterior muscle in the mouse hindlimb, followed by electroporation of the muscle with needle electrodes. We observed VEGF levels significantly above background 40 days after injection in animals that received phiC31 integrase and the VEGF plasmid. Site-specific integration of plasmid DNA in the chromosomes of muscle tissue was verified by polymerase chain reaction at a common integration site. These results suggest the possible utility of the phiC31 integrase system to treat ischemic disease.

Long-term expression of human coagulation factor VIII in a tolerant mouse model using the φC31 integrase system.

We generated a mouse model for hemophilia A that combines a homozygous knockout for murine factor VIII (FVIII) and a homozygous addition of a mutant human FVIII (hFVIII). The resulting mouse, having no detectable FVIII protein or activity and tolerant to hFVIII, is useful for evaluating FVIII gene-therapy protocols. This model was used to develop an effective gene-therapy strategy using the φC31 integrase to mediate permanent genomic integration of an hFVIII cDNA deleted for the B-domain. Various plasmids encoding φC31 integrase and hFVIII were delivered to the livers of these mice by using hydrodynamic tail-vein injection. Long-term expression of therapeutic levels of hFVIII was observed over a 6-month time course when an intron was included in the hFVIII expression cassette and wild-type φC31 integrase was used. A second dose of the hFVIII and integrase plasmids resulted in higher long-term hFVIII levels, indicating that incremental doses were beneficial and that a second dose of φC31 integrase was tolerated. We observed a significant decrease in the bleeding time after a tail-clip challenge in mice treated with plasmids expressing hFVIII and φC31 integrase. Genomic integration of the hFVIII expression plasmid was demonstrated by junction PCR at a known hotspot for integration in mouse liver. The φC31 integrase system provided a nonviral method to achieve long-term FVIII gene therapy in a relevant mouse model of hemophilia A.

Kinetics and longevity of ΦC31 integrase in mouse liver and cultured cells.

The ΦC31 integrase system provides genomic integration of plasmid DNA that may be useful in gene therapy. For example, the ΦC31 system has been used in combination with hydrodynamic injection to achieve long-term expression of factor IX in mouse liver. However, a concern is that prolonged expression of ΦC31 integrase within cells could potentially stimulate chromosome rearrangements or an immune response. Western blot and immunofluorescence analyses were performed to investigate the duration of ΦC31 integrase expression in mouse liver. Integrase was expressed within 2 to 3 hr after hydrodynamic injection of a plasmid expressing ΦC31 integrase. Expression peaked between 8 and 16 hr and fell to background levels by 24-48 hr postinjection. Analysis of the amount of integrase plasmid DNA present in the liver over time suggested that the brief period of integrase expression could largely be accounted for by rapid loss of the bulk of the plasmid DNA, as well as by silencing of plasmid expression. PCR analysis of integration indicated that ΦC31 integrase carried out genomic integration of a codelivered attB-containing plasmid by 3 hr after plasmid injection. Integrase was expressed for longer times and at higher levels in transfected cultured cells compared with liver. Inhibitor studies suggested that the enzyme had a short half-life and was degraded by the 26S proteasome. The short duration of integrase expression in liver and rapid integration reaction appear to be features favorable for use in gene therapy.

Impact of hydrodynamic injection and phiC31 integrase on tumor latency in a mouse model of MYC-induced hepatocellular carcinoma.

BACKGROUND: Hydrodynamic injection is an effective method for DNA delivery in mouse liver and is being translated to larger animals for possible clinical use. Similarly, phiC31 integrase has proven effective in mediating long-term gene therapy in mice when delivered by hydrodynamic injection and is being considered for clinical gene therapy applications. However, chromosomal aberrations have been associated with phiC31 integrase expression in tissue culture, leading to questions about safety. METHODOLOGY/PRINCIPAL FINDINGS: To study whether hydrodynamic delivery alone, or in conjunction with delivery of phiC31 integrase for long-term transgene expression, could facilitate tumor formation, we used a transgenic mouse model in which sustained induction of the human C-MYC oncogene in the liver was followed by hydrodynamic injection. Without injection, mice had a median tumor latency of 154 days. With hydrodynamic injection of saline alone, the median tumor latency was significantly reduced, to 105 days. The median tumor latency was similar, 106 days, when a luciferase donor plasmid and backbone plasmid without integrase were administered. In contrast, when active or inactive phiC31 integrase and donor plasmid were supplied to the mouse liver, the median tumor latency was 153 days, similar to mice receiving no injection. CONCLUSIONS/SIGNIFICANCE: Our data suggest that phiC31 integrase does not facilitate tumor formation in this C-MYC transgenic mouse model. However, in groups lacking phiC31 integrase, hydrodynamic injection appeared to contribute to C-MYC-induced hepatocellular carcinoma in adult mice. Although it remains to be seen to what extent these findings may be extrapolated to catheter-mediated hydrodynamic delivery in larger species, they suggest that caution should be used during translation of hydrodynamic injection to clinical applications.

Human, viral or mutant human IL-10 expressed after local adenovirus-mediated gene transfer are equally effective in ameliorating disease pathology in a rabbit knee model of antigen-induced arthritis.

IL-10 is a Th2 cytokine important for inhibiting cell-mediated immunity while promoting humoral responses. Human IL-10 (hIL-10) has anti-inflammatory, immunosuppressive as well as immunostimulatory characteristics, whereas viral IL-10 (vIL-10), a homologue of hIL-10 encoded by Epstein Barr virus (EBV), lacks several immunostimulatory functions. The immunostimulatory characteristic of hIL-10 has been attributed to a single amino acid, isoleucine at position 87, which in vIL-10 is alanine. A mutant hIL-10 in which isoleucine has been substituted (mut.hIL-10) is biologically active with only immunosuppressive, but not immunostimulatory, functions, making it a potentially superior therapeutic for inflammatory diseases. To compare the efficacy of mut.hIL-10 with hIL-10 and vIL-10 in blocking the progression of rheumatoid arthritis, we used replication defective adenoviral vectors to deliver intra-articularly the gene encoding hIL-10, vIL-10 or mut.hIL-10 to antigen-induced arthritic (AIA) knee joints in rabbits. Intra-articular expression of hIL-10, vIL-10, and mut.hIL-10 resulted in significant improvement of the pathology in the treated joints to similar levels. These observed changes included a significant reduction in intra-articular leukocytosis and the degree of synovitis, as well as normalization of cartilage matrix metabolism. Our results suggest that hIL-10, vIL-10, and mut.hIL-10 are all equally therapeutic in the rabbit AIA model for treating disease pathology.

PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints.

BACKGROUND: Gene transfer to synovium in joints has been shown to be an effective approach for treating pathologies associated with rheumatoid arthritis (RA) and related joint disorders. However, the efficiency and duration of gene delivery has been limiting for successful gene therapy for arthritis. The transient gene expression that often accompanies non-viral gene delivery can be prolonged by integration of vector DNA into the host genome. We report a novel approach for non-viral gene therapy to joints that utilizes phage phiC31 integrase to bring about unidirectional genomic integration. METHODS: Rabbit and human synovial cells were co-transfected with a plasmid expressing phiC31 integrase and a plasmid containing the transgene and an attB site. Cells were cultured with or without G418 selection and the number of neo-resistant colonies or eGFP cells determined, respectively. Plasmid rescue, PCR query, and DNA sequence analysis were performed to reveal integration sites in the rabbit and human genomes. For in vivo studies, attB-reporter gene plasmids and a plasmid expressing phiC31 integrase were intra-articularly injected into rabbit knees. Joint sections were used for histological analysis of beta-gal expression, and synovial cells were isolated to measure luciferase expression. RESULTS: We demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the transgene and attB increased the frequency of transgene expression in rabbit synovial fibroblasts and primary human RA synoviocytes. Plasmid rescue and DNA sequence analysis of plasmid-chromosome junctions revealed integration at endogenous pseudo attP sequences in the rabbit genome, and PCR query detected integration at previously characterized integration sites in the human genome. Significantly higher levels of transgene expression were detected in vivo in rabbit knees after intra-articular injection of attB-reporter gene plasmids and a plasmid expressing phiC31 integrase. CONCLUSION: The ability of phiC31 integrase to facilitate genomic integration in synovial cells and increase transgene expression in the rabbit synovium suggests that, in combination with more efficient DNA delivery methods, this integrase system could be beneficial for treatment of rheumatoid arthritis and other joint disorders.

A diversity of serine phage integrases mediate site-specific recombination in mammalian cells.

This study evaluated the ability of five serine phage integrases, from phages A118, U153, Bxb1, phiFC1, and phiRV1, to mediate recombination in mammalian cells. Two types of recombination were investigated, including the ability of an integrase to mediate recombination between its own phage att sites in the context of a mammalian cell and the ability of an integrase to perform genomic integration pairing a phage att site with an endogenous mammalian sequence. We demonstrated that the A118 integrase mediated precise intra-molecular recombination of a plasmid containing its attB and attP sites at a frequency of approximately 50% in human cells. The closely related U153 integrase also performed efficient recombination in human cells on a plasmid containing the attB and attP sites of A118. The integrases from phages Bxb1, phiFC1, and phiRV1 carried out such recombination at their attB and attP sites at frequencies ranging from 11 to 75%. Furthermore, the A118 integrase mediated recombination between its attP site on a plasmid and pseudo attB sites in the human genome, i.e. native sequences with partial identity to attB. Fifteen such A118 pseudo att sites were analyzed, and a consensus recognition site was identified. The other integrases did not mediate integration at genomic sequences at a frequency above background. These site-specific integrases represent valuable new tools for manipulating eukaryotic genomes.

Exosomes derived from IL-10-treated dendritic cells can suppress inflammation and collagen-induced arthritis.

We have demonstrated previously that local, adenoviral-mediated gene transfer of viral IL-10 to a single joint of rabbits and mice with experimental arthritis can suppress disease in both the treated and untreated contralateral joints. This contralateral effect is mediated in part by APCs able to traffic from the treated joint to lymph nodes as well as to untreated joints. Moreover, injection of dendritic cells (DC) genetically modified to express IL-4 or Fas ligand was able to reverse established murine arthritis. To examine the ability of exosomes derived from immunosuppressive DCs to reduce inflammation and autoimmunity, murine models of delayed-type hypersensitivity and collagen-induced arthritis were used. In this study, we demonstrate that periarticular administration of exosomes purified from either bone marrow-derived DCs transduced ex vivo with an adenovirus expressing viral IL-10 or bone marrow-derived DCs treated with recombinant murine IL-10 were able to suppress delayed-type hypersensitivity responses within injected and untreated contralateral joints. In addition, the systemic injection of IL-10-treated DC-derived exosomes was able suppress the onset of murine collagen-induced arthritis as well as reduce severity of established arthritis. Taken together, these data suggest that immature DCs are able to secrete exosomes that are involved in the suppression of inflammatory and autoimmune responses. Thus DC-derived exosomes may represent a novel, cell-free therapy for the treatment of autoimmune diseases.