Zhangyong Wei

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

Deficiency of SPAG16L Causes Male Infertility Associated with Impaired Sperm Motility

Abstract The axonemes of cilia and flagella contain a “9+2” structure of microtubules and associated proteins. Proteins associated with the central doublet pair have been identified in Chlamydomonas that result in motility defects when mutated. The murine orthologue of the Chlamydomonas PF20 gene, sperm-associated antigen 16 (Spag16), encodes two proteins of Mr ∼71 × 103 (SPAG16L) and Mr ∼35 × 103 (SPAG16S). In sperm, SPAG16L is found in the central apparatus of the axoneme. To determine the function of SPAG16L, gene targeting was used to generate mice lacking this protein but still expressing SPAG16S. Mutant animals were viable and showed no evidence of hydrocephalus, lateralization defects, sinusitis, bronchial infection, or cystic kidneys—symptoms typically associated with ciliary defects. However, males were infertile with a lower than normal sperm count. The sperm had marked motility defects, even though ultrastructural abnormalities of the axoneme were not evident. In addition, the testes of some nullizygous animals showed a spermatogenetic defect, which consisted of degenerated germ cells in the seminiferous tubules. We conclude that SPAG16L is essential for sperm flagellar function. The sperm defect is consistent with the motility phenotype of the Pf20 mutants of Chlamydomonas, but morphologically different in that the mutant algal axoneme lacks the central apparatus.

Safety and Efficacy of Subretinal Readministration of a Viral Vector in Large Animals to Treat Congenital Blindness

After successful gene therapy to correct retinal degeneration in one eye, treatment of the second eye is safe and effective—even when immunity to the vector is present—in nonhuman primates and dogs. Both Sides Now Blind and deaf from birth, Helen Keller—whose life is the subject of the drama The Miracle Worker—once remarked that “the world is full of suffering; it is also full of overcoming.” Ms. Keller knew something about rising above one’s circumstances—despite her dual disability, she became a renowned author, activist, and lecturer. Last year, through a ground-breaking clinical trial, a research team from The Children’s Hospital of Philadelphia (CHOP) and the University of Pennsylvania helped a group of children to overcome their near-complete blindness by a different route—a gene therapy regimen that replaced a mutated gene and partially restored visual function in one eye of all 12 patients. Now, this same team of researchers has taken an essential step toward the ability to offer these patients the same sight-restoring treatment for their second eyes. These trial participants had all inherited a specific type of Leber’s congenital amaurosis (LCA), a class of progressive retinal degenerative conditions that can be detected in infancy and results in complete blindness by the age of 40. The loss of photosensitive retinal cells characteristic of this class of diseases stems from mutations in one of 14 genes. The LCA-RPE65 patients in the CHOP trial all had aberrant copies of the RPE65 gene, which encodes an enzyme in the retinal pigment epithelium whose function is a crucial component of the visual cycle. Therefore, surgeons delivered a wild-type copy of RPE65 housed in an adeno-associated virus vector (AAV2-hRPE65v2) into one of the eyes of each of these trial subjects. As one might imagine, the sight-restoring success of this gene therapy trial has patients clamoring for more—more treatment, that is, of their second eyes. But first, researchers needed to assess whether a second treatment would be safe and effective. Specifically, scientists investigated whether subjects who developed neutralizing antibodies (NAbs) directed against the AAV vector used in the first round of gene therapy would benefit from a second round or whether the anti-AAV NAbs would impede gene transfer. This week’s issue of Science Translational Medicine reports the results of Amado et al. on the immunological and functional consequences of serial subretinal readministration of the RPE65-carrying AAV vector. The authors performed their studies in two large animal models—a canine model of LCA-RPE65 and nonhuman primates without eye disease. Both animals sport eyes similar in size to those of humans, and all primates have macula, a specialized region of the retina responsible for central vision. Also, because nonhuman primates can be infected with wild-type AAV and develop antibodies to viral components, these animals likely give researchers a window into the human immune response that manifests upon vector injection. The authors show that the readministration treatment is safe and effective in both models, even in animals that display preexisting immunity to the vector. These results suggest that a patient who has undergone AAV2-hRPE65v2—mediated gene therapy in one eye may indeed enjoy success from a second surgery. Furthermore, scientists had routinely excluded from clinical trials patients who already carried NAbs to AAVs in their sera. And, as Amado et al. also show, this crowd encompasses a substantial portion of the human population, a number that increases with age. Now, LCA-RPE65 patients with antibodies to AAV may be eligible for gene therapy. This study thus represents an essential step in the further translation of gene therapy for LCA—and a reason for celebration in the gene therapy research community. Leber’s congenital amaurosis (LCA) is a group of severe inherited retinal degenerations that are symptomatic in infancy and lead to total blindness in adulthood. Recent clinical trials using recombinant adeno-associated virus serotype 2 (rAAV2) successfully reversed blindness in patients with LCA caused by RPE65 mutations after one subretinal injection. However, it was unclear whether treatment of the second eye in the same manner would be safe and efficacious, given the potential for a complicating immune response after the first injection. Here, we evaluated the immunological and functional consequences of readministration of rAAV2-hRPE65v2 to the contralateral eye using large animal models. Neither RPE65-mutant (affected; RPE65−/−) nor unaffected animals developed antibodies against the transgene product, but all developed neutralizing antibodies against the AAV2 capsid in sera and intraocular fluid after subretinal injection. Cell-mediated immune responses were benign, with only 1 of 10 animals in the study developing a persistent T cell immune response to AAV2, a response that was mediated by CD4+ T cells. Sequential bilateral injection caused minimal inflammation and improved visual function in affected animals. Thus, subretinal readministration of rAAV2 in animals is safe and effective, even in the setting of preexisting immunity to the vector, a parameter that has been used to exclude patients from gene therapy trials.

Dissecting the Axoneme Interactome The Mammalian Orthologue of Chlamydomonas PF6 Interacts with Sperm-Associated Antigen 6, The Mammalian Orthologue of Chlamydomonas PF16

The axoneme central apparatus is thought to control flagellar/ciliary waveform and maintain the structural integrity of the axoneme, but proteins involved in these processes have not been fully elucidated. Moreover the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule. Mutations in the Chlamydomonas PF6 gene result in flagellar paralysis. We characterized human and murine orthologues of PF6. The murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia, and the PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that a portion of PF6 associates with the mammalian orthologue of Chlamydomonas PF16 (sperm-associated antigen 6 (SPAG6)), another central apparatus protein that is localized to the C1 microtubule in algae. A fragment of PF6 corresponding to the PF6 domain that interacts with SPAG6 in yeast two-hybrid assays and colocalizes with SPAG6 in transfected cells was missing from epididymal sperm of SPAG6-deficient mice. SPAG6 binds to the mammalian orthologue of PF20, which in Chlamydomonas is located in bridges connecting the C2 and C1 microtubules. Thus, PF6, SPAG6, and PF20 form a newly identified network that links together components of the axoneme central apparatus and presumably participates in its dynamic regulation of ciliary and flagellar beat.

Dissecting the axoneme interactome: The mammalian orthologue of chlamydomonas PF6 interacts with SPAG6, the mammalian orthologue of chlamydomonas PF16

The axoneme central apparatus is thought to control flagellar/ciliary waveform and maintain the structural integrity of the axoneme, but proteins involved in these processes have not been fully elucidated. Moreover the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule. Mutations in the Chlamydomonas PF6 gene result in flagellar paralysis. We characterized human and murine orthologues of PF6. The murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia, and the PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that a portion of PF6 associates with the mammalian orthologue of Chlamydomonas PF16 (sperm-associated antigen 6 (SPAG6)), another central apparatus protein that is localized to the C1 microtubule in algae. A fragment of PF6 corresponding to the PF6 domain that interacts with SPAG6 in yeast two-hybrid assays and colocalizes with SPAG6 in transfected cells was missing from epididymal sperm of SPAG6-deficient mice. SPAG6 binds to the mammalian orthologue of PF20, which in Chlamydomonas is located in bridges connecting the C2 and C1 microtubules. Thus, PF6, SPAG6, and PF20 form a newly identified network that links together components of the axoneme central apparatus and presumably participates in its dynamic regulation of ciliary and flagellar beat.