Caspase Inhibition with XIAP as an Adjunct to AAV Vector Gene-Replacement Therapy: Improving Efficacy and Prolonging the Treatment Window

AAV-mediated gene therapy in the rd10 mouse, with retinal degeneration caused by mutation in the rod cyclic guanosine monophosphate phosphodiesterase β-subunit (PDEβ) gene, produces significant, but transient, rescue of photoreceptor structure and function. This study evaluates the ability of AAV-mediated delivery of X-linked inhibitor of apoptosis (XIAP) to enhance and prolong the efficacy of PDEβ gene-replacement therapy.Rd10 mice were bred and housed in darkness. Two groups of animals were generated: Group 1 received sub-retinal AAV5-XIAP or AAV5-GFP at postnatal age (P) 4 or 21 days; Group 2 received sub-retinal AAV5-XIAP plus AAV5- PDEβ, AAV5-GFP plus AAV5- PDEβ, or AAV- PDEβ alone at age P4 or P21. Animals were maintained for an additional 4 weeks in darkness before being moved to a cyclic-light environment. A subset of animals from Group 1 received a second sub-retinal injection of AAV8-733-PDEβ two weeks after being moved to the light. Histology, immunohistochemistry, Western blots, and electroretinograms were performed at different times after moving to the light.Injection of AAV5-XIAP alone at P4 and 21 resulted in significant slowing of light-induced retinal degeneration, as measured by outer nuclear thickness and cell counts, but did not result in improved outer segment structure and rhodopsin localization. In contrast, co-injection of AAV5-XIAP and AAV5-PDEβ resulted in increased levels of rescue and decreased rates of retinal degeneration compared to treatment with AAV5-PDEβ alone. Mice treated with AAV5-XIAP at P4, but not P21, remained responsive to subsequent rescue by AAV8-733-PDEβ when injected two weeks after moving to a light-cycling environment.Adjunctive treatment with the anti-apoptotic gene XIAP confers additive protective effect to gene-replacement therapy with AAV5-PDEβ in the rd10 mouse. In addition, AAV5-XIAP, when given early, can increase the age at which gene-replacement therapy remains effective, thus effectively prolonging the window of opportunity for therapeutic intervention

Acute effects of hemodialysis on nitrite and nitrate: potential cardiovascular implications in dialysis patients

Cardiovascular mortality in dialysis patients remains a serious problem. It is 10 to 20 times higher than in the general population. No molecular mechanism has been proven to explain this increased mortality, although nitric oxide (NO) has been implicated. The objective of our study was to determine the extent of the removal of the NO congeners nitrite and nitrate from plasma and saliva by hemodialysis, as this might disrupt physiological NO bioactivity and help explain the health disparity in dialysis patients. Blood and saliva were collected at baseline from patients on dialysis and blood was collected as it exited the dialysis unit. Blood and saliva were again collected after 4-5h of dialysis. In the 27 patients on dialysis, baseline plasma nitrite and nitrate by HPLC were 0.21±0.03 and 67.25±14.68 μM, respectively. Blood immediately upon exit from the dialysis unit had 57% less nitrite (0.09±0.03 μM; P=0.0008) and 84% less nitrate (11.04 μM; P=0.0003). After 4-5h of dialysis, new steady-state plasma levels of nitrite and nitrate were significantly lower than baseline, 0.09±0.01 μM (P=0.0002) and 16.72±2.27 μM (P=0.001), respectively. Dialysis also resulted in a significant reduction in salivary nitrite (232.58±75.65 to 25.77±10.88 μM; P=0.01) and nitrate (500.36±154.89 to 95.08±24.64 μM; P=0.01). Chronic and persistent depletion of plasma and salivary nitrite and nitrate probably reduces NO bioavailability and may explain in part the increased cardiovascular mortality in the dialysis patient

Phenotypic Conservation in Patients With X-Linked Retinitis Pigmentosa Caused by RPGR Mutations

ImportanceFor patients with X-linked retinitis pigmentosa and clinicians alike, phenotypic variability can be challenging because it complicates counseling regarding patients' likely visual prognosis.ObjectiveTo evaluate the clinical findings from patients with X-linked retinitis pigmentosa with 13 distinct RPGR mutations and assess for phenotypic concordance or variability.DesignRetrospective medical record review of data collected from 1985 to 2011.SettingKellogg Eye Center, University of Michigan.PatientsA total of 42 patients with X-linked retinitis pigmentosa with mutations in RPGR. Age at first visit ranged from 4 to 53 years, with follow-up ranging from 1 to 11 visits (median follow-up time, 5.5 years; range, 1.4-32.7 years, for 23 patients with >1 visit).Main outcomes and measuresClinical data assessed for concordance included visual acuity (VA), Goldmann visual fields (GVFs), and full-field electroretinography (ERG). Electroretinography phenotype (cone-rod vs rod-cone dysfunction) was defined by the extent of photopic vs scotopic abnormality. Qualitative GVF phenotype was determined by the GVF pattern, where central or peripheral loss suggested cone or rod dysfunction, respectively. Goldmann visual fields were also quantified and compared among patients.ResultsEach mutation was detected in 2 or more related or unrelated patients. Five mutations in 11 patients displayed strong concordance of VA, while 4 mutations in 16 patients revealed moderate concordance of VA. A definitive cone-rod or rod-cone ERG pattern consistent among patients was found in 6 of 13 mutations (46.2%); the remaining mutations were characterized by patients demonstrating both phenotypes or who had limited data or nonrecordable ERG values. Concordant GVF phenotypes (7 rod-cone pattern vs 4 cone-rod pattern) were seen in 11 of 13 mutations (84.6%). All 6 mutations displaying a constant ERG pattern within the mutation group revealed a GVF phenotype consistent with the ERG findings.Conclusions and relevanceWhile VA and ERG phenotypes are concordant in only some patients carrying identical mutations, assessment of GVF phenotypes revealed stronger phenotypic conservation. Phenotypic concordance is important for establishing proper counseling of patients diagnosed as having X-linked retinitis pigmentosa, as well as for establishing accurate patient selection and efficacy monitoring in therapeutic trials

Phenotypic Conservation in Patients With X-Linked Retinitis Pigmentosa Caused by RPGR

IMPORTANCE: For patients with X-linked retinitis pigmentosa and clinicians alike, phenotypic variability can be challenging because it complicates counseling regarding patients’ likely visual prognosis. OBJECTIVE: To evaluate the clinical findings from patients with X-linked retinitis pigmentosa with 13 distinct RPGR mutations and assess for phenotypic concordance or variability. DESIGN: Retrospective medical record review of data collected from 1985 to 2011. SETTING: Kellogg Eye Center, University of Michigan. PATIENTS: A total of 42 patients with X-linked retinitis pigmentosa with mutations in RPGR. Age at first visit ranged from 4 to 53 years, with follow-up ranging from 1 to 11 visits (median follow-up time, 5.5 years; range, 1.4-32.7 years, for 23 patients with >1 visit). MAIN OUTCOMES AND MEASURES: Clinical data assessed for concordance included visual acuity (VA), Goldmann visual fields (GVFs), and full-field electroretinography (ERG). Electroretinography phenotype (cone-rod vs rod-cone dysfunction) was defined by the extent of photopic vs scotopic abnormality. Qualitative GVF phenotype was determined by the GVF pattern, where central or peripheral loss suggested cone or rod dysfunction, respectively. Goldmann visual fields were also quantified and compared among patients. RESULTS: Each mutation was detected in 2 or more related or unrelated patients. Five mutations in 11 patients displayed strong concordance of VA, while 4 mutations in 16 patients revealed moderate concordance of VA. A definitive cone-rod or rod-cone ERG pattern consistent among patients was found in 6 of 13 mutations (46.2%); the remaining mutations were characterized by patients demonstrating both phenotypes or who had limited data or nonrecordable ERG values. Concordant GVF phenotypes (7 rod-cone pattern vs 4 cone-rod pattern) were seen in 11 of 13 mutations (84.6%). All 6 mutations displaying a constant ERG pattern within the mutation group revealed a GVF phenotype consistent with the ERG findings. CONCLUSIONS AND RELEVANCE: While VA and ERG phenotypes are concordant in only some patients carrying identical mutations, assessment of GVF phenotypes revealed stronger phenotypic conservation. Phenotypic concordance is important for establishing proper counseling of patients diagnosed as having X-linked retinitis pigmentosa, as well as for establishing accurate patient selection and efficacy monitoring in therapeutic trials

ERG analysis of retinal function in <i>rd10</i> mice treated by co-injection of AAV5-XIAP and AAV5-PDEβ.

<p><b>A, C.</b> Scotopic B wave responses of treated mice at 0, 1, 2, 3 and 6 weeks after being moved to the light-environment were normalized to the scotopic B wave of untreated retinas at time 0. Responses in mice treated with AAV5-XIAP plus AAV5-PDEβ were greater and maintained longer that those of mice treated with AAV5-GFP plus AAV5-PDEβ. The absence of rescue by AAV5-GFP alone excludes the possibility that rescue results from the surgical procedure alone or exposure to intact vector. Similar outcomes were obtained in mice treated at P4 and P21. <b>B, D.</b> Representative ERG tracings of <i>rd10</i> mice treated with AAV5-XIAP plus AAV5-PDEβ, AAV5-GFP plus AAV5-PDEβ, or untreated, at 0, 1, 2, 3 and 6 weeks after being moved to the light environment. The response of untreated retinas at t = 0 was larger than the response of treated retinas, presumably because of some deleterious effects caused by the transient retinal detachment that necessarily accompanies sub-retinal vector injection. Untreated retinas, however, quickly lose all ERG responses, with no detectable B wave left by 1 week after moving into the light.</p

Co-transduction of <i>rd10</i> retinas with AAV5-PDEβ and AAV5-XIAP at P4 and P21.

<p><b>A.</b> Western blot of PDEβ expression in AAV5-PDEβ transduced <i>rd10</i> retinas compared to wild type (C57) retinas. <b>B.</b> Confocal imaging of isolated rod outer segments from AAV5-PDEβ transduced <i>rd10</i> retinas showing expression of PDEβ (red). <b>C–D.</b> Co-treatment of <i>rd10</i> retinas with AAV5-XIAP and AAV5-PDEβ showing improved survival of photoreceptor cells compared to retinas treated with AAV5-PDEβ alone or in combination with AAV5-GFP. <b>C.</b> Co-injection at PN4. <b>D.</b> Co-injection at PN21.</p

Sequential treatment of <i>rd10</i> mice with AAV5-XIAP and AAV8-733-PDEβ.

<p>Dark-reared <i>rd10</i> mice were treated with AAV5-XIAP by sub-retinal injection at age P4 or P21, and then maintained in darkness for 4 weeks before being moved to the light environment. After 2 weeks in the light, the mice were treated with AAV8-733-PDEβ by sub-retinal injection, and then evaluated at the times indicated. Control eyes received only the AAV8-733-PDEβ treatment two weeks after being moved to the light environment. <b>A.</b> Thickness measurements show slowing of ONL cell losses by treatment of <i>rd10</i> mice with AAV5-XIAP alone. In animals treated with AAV5-XIAP at age P4, but not at P21, further losses of ONL thickness were halted by the subsequent administration of AAV8-733-PDEβ. <b>B.</b> Western blots of retinas sequentially injected with AAV5-XIAP and AAV8-733-PDEβ, and examined 4 weeks later, show expression of recombinant XIAP and PDEβ in transduced retinas, and preservation of the expression of endogenous PDEa, rhodopsin (Rho), transducin and guanylate cyclase 1 (GC1). <b>C.</b> Histology of retinas sequentially injected with AAV5-XIAP and AAV8-733-PDEβ, and examined 4 weeks later, shows localization of XIAP (green – via staining of HA-tag) to inner segments and PDEβ (red) and rhodopsin (purple) to outer segments. Outer segments appear to be shortened, and there is mislocalization of PDEβ and rhodopsin to the inner segments.</p

Retinal histology of AAV5-PDEβ transduced <i>rd10</i>retinas.

<p><b>Top Panel.</b> Sub-retinal injection of AAV5-PDEβ co-treated with AAV5-GFP resulted in significant preservation of outer nuclear layer and inner and outer segment morphology. (GFP, green; PDEβ, red). <b>Bottom Panel.</b> Co-injection with AAV5-XIAP and AAV5-PDEβ shows improved preservation of retinal structures. XIAP (green – via staining of HA-tag) and PDEβ (red) localized to inner and outer segments, respectively.</p

Effects of subretinal injection of AAV5-XIAP, or a control AAV5-GFP construct, on <i>rd10</i> mice treated at P4 and dark-reared for 4 weeks post treatment before being moved to the light environment.

<p><b>A.</b> Western-blot analysis shows expression of recombinant XIAP. <b>B.</b> Caspase 3 activity assayed 24 hours after transfer from the dark-rearing to the light environment shows AAV5-XIAP reduced caspase 3 activity levels in untreated <i>rd10</i> retinas to levels found in control C57 mice. <b>C.</b> Retinal histology at 3 weeks after moving to the light environment shows extensive degeneration of the ONL in untreated (a,d) and AAV5-GFP treated retinas (b,e; green shows localization of GFP), and significant preservation of ONL thickness and cell counts in the AAV5-XIAP treated retinas (c,f; green showing localization of XIAP by staining of HA-tag).</p

Retinal function and protein expression in 1 and 2 month-old dark-reared <i>rd10</i> mice.

<p><b>A.</b> Normalized scotopic ERG B-wave responses showing age-dependent loss-of-function under conditions where ONL cell counts were preserved. <b>B.</b> Western blots of PDEα, transducin, rhodopsin (Rho) and guanylate cyclase 1 (GC1) expression showing significant age-dependent losses occurring in the dark, and catastrophic losses occurring 24 hours after moving to the light environment.</p