Fluorescence studies on the bases of the nucleic acids in solution at room temperature

Fluorescence of adenine, guanine, thymine, cytosine, and uracil has been detected at room temperature in neutral aqueous solution using a digital signal accumulation technique and right angle fluorescence detection from solutions with absorbances between 0. 4 and 0. 7. The quantum yields are respectively 2.6 x 10⁻⁴, 3.0 x 10⁻⁴, 1 x 10⁻⁴ 0.8 x 10⁻⁴, and 0.5 x 10⁻⁴ when excited at their low energy absorption maxima. Singlet lifetimes calculated from these yields may mean a recalculation of excited state processes occurring in DNA which were previously based on low temperature data. Corrected relative emission spectra are presented and compared with reported low temperature data. The corrected relative excitation spectra all show significant differences from the absorption spectrum when both are determined under identical conditions of concentration and spectral bandwidth on the same instrument. This behavior is discussed in terms J. of a) n-π and π-π states, b) emission from a minor tautomer and c) kinetics of competing deactivation processes. From a development of c), rate constants for processes occurring from the 0' level are calculated for intersystem crossing, internal conversion, and (singlet) photochemical reaction in thymine and uracil. From solvent induced shifts of absorption and fluorescence spectra, the excited state dipole moment of thymine μe = 8. 0 ± 2. 20) and the π-π* nature of the transitions responsible for fluorescence are deduced. Consideration of the singlet lifetime of thymine in relation to the rates of excited state process implies that fluorescence may occur from a Franck-Condon excited state and this is discussed in terms of possible solute-solvent interactions. A study of the pH effects on the spectral shape, magnitude, and polarization of thymine emission concludes that the emitting states of J. ionized thymine are of a π-π* nature , that fluorescence may arise within the # 1, 2,4, 5, and 6 π- conjugated system in the thymine molecule, and that excited state ionizations do not occur in the fluorescent forms of thymine. These deductions are discussed in relation to DNA excited state processes

Reply to Townes-Anderson: RPE65 Gene Therapy Does Not Alter the Natural History of Retinal Degeneration

We appreciate the interest shown by TownesAnderson in our article examining the natural history of retinal degeneration in Leber congenital amaurosis caused by retinal pigment epithelium-specific protein 65kDa (RPE65) mutations and evaluating the consequences of gene augmentation therapy. Townes-Anderson’s remarks focused on the final phrase of the last sentence of the Discussion of our article. In the full sentence, we suggested that in the future, agents to reduce cell death could be delivered in combination with a more advanced version of the gene augmentation therapy that reaches not only remaining rods and extrafoveal cones but also foveal cone photoreceptors

rAAV2/5 Gene-Targeting to Rods: Dose-Dependent Efficiency and Complications Associated With Different Promoters

A prerequisite for using corrective gene therapy to treat humans with inherited retinal degenerative diseases that primarily affect rods is to develop viral vectors that target specifically this population of photoreceptors. The delivery of a viral vector with photoreceptor tropism coupled with a rod-specific promoter is likely to be the safest and most efficient approach to target expression of the therapeutic gene to rods. Three promoters that included a fragment of the proximal mouse opsin promoter (mOP), the human G-protein-coupled receptor protein kinase 1 promoter (hGRK1), or the cytomegalovirus immediate early enhancer combined with the chicken β actin proximal promoter CBA were evaluated for their specificity and robustness in driving GFP reporter gene expression in rods, when packaged in a recombinant adeno-associated viral vector of serotype 2/5 (AAV2/5), and delivered via subretinal injection to the normal canine retina. Photoreceptor-specific promoters (mOP, hGRK1) targeted robust GFP expression to rods, whereas the ubiquitously expressed CBA promoter led to transgene expression in the retinal pigment epithelium, rods, cones and rare Müller, horizontal and ganglion cells. Late onset inflammation was frequently observed both clinically and histologically with all three constructs when the highest viral titers were injected. Cone loss in the injected regions of the retinas that received the highest titers occurred with both the hGRK1 and CBA promoters. Efficient and specific rod transduction, together with preservation of retinal structure was achieved with both mOP and hGRK1 promoters when viral titers in the order of 1011 vg ml–1 were used

Rapid, widespread transduction of the murine myocardium using self-complementary Adeno-associated virus

Adeno-associated virus (AAV) has shown great promise as a gene transfer vector. However, the incubation time needed to attain significant levels of gene expression is often too long for some clinical applications. Self-complementary AAV (scAAV) enters the cell as double stranded DNA, eliminating the step of second-strand synthesis, proven to be the rate-limiting step for gene expression of single-stranded AAV (ssAAV). The aim of this study was to compare the efficiency of these two types of AAV vectors in the murine myocardium. Four day old CD-1 mice were injected with either of the two AAV constructs, both expressing GFP and packaged into the AAV1 capsid. The animals were held for 4, 6, 11 or 21 days, after which they were euthanized and their hearts were excised. Serial sections of the myocardial tissue were used for real-time PCR quantification of AAV genome copies and for confocal microscopy. Although we observed similar numbers of AAV genomes at each of the different time points present in both the scAAV and the ssAAV infected hearts, microscopic analysis showed expression of GFP as early as 4 days in animals injected with the scAAV, while little or no expression was observed with the ssAAV constructs until day 11. AAV transduction of murine myocardium is therefore significantly enhanced using scAAV constructs

Recombinant AAV-Mediated \u3cem\u3eBEST1\u3c/em\u3e Transfer to the Retinal Pigment Epithelium: Analysis of Serotype-Dependent Retinal Effects

Mutations in the BEST1 gene constitute an underlying cause of juvenile macular dystrophies, a group of retinal disorders commonly referred to as bestrophinopathies and usually diagnosed in early childhood or adolescence. The disease primarily affects macular and paramacular regions of the eye leading to major declines in central vision later in life. Currently, there is no cure or surgical management for BEST1-associated disorders. The recently characterized human disease counterpart, canine multifocal retinopathy (cmr), recapitulates a full spectrum of clinical and molecular features observed in human bestrophinopathies and offers a valuable model system for development and testing of therapeutic strategies. In this study, the specificity, efficiency and safety of rAAV-mediated transgene expression driven by the human VMD2 promoter were assessed in wild-type canine retinae. While the subretinal delivery of rAAV2/1 vector serotype was associated with cone damage in the retina when BEST1 and GFP were co-expressed, the rAAV2/2 vector serotype carrying either GFP reporter or BEST1 transgene under control of human VMD2 promoter was safe, and enabled specific transduction of the RPE cell monolayer that was stable for up to 6 months post injection. These encouraging studies with the rAAV2/2 vector lay the groundwork for development of gene augmentation therapy for human bestrophinopathies

Cone Phosphodiesterase-6γ’ Subunit Augments Cone PDE6 Holoenzyme Assembly and Stability in a Mouse Model Lacking Both Rod and Cone PDE6 Catalytic Subunits

Rod and cone phosphodiesterase 6 (PDE6) are key effector enzymes of the vertebrate phototransduction pathway. Rod PDE6 consists of two catalytic subunits PDE6α and PDE6β and two identical inhibitory PDE6γ subunits, while cone PDE6 is composed of two identical PDE6α’ catalytic subunits and two identical cone-specific PDE6γ’ inhibitory subunits. Despite their prominent function in regulating cGMP levels and therefore rod and cone light response properties, it is not known how each subunit contributes to the functional differences between rods and cones. In this study, we generated an rd10/cpfl1 mouse model lacking rod PDE6β and cone PDE6α’ subunits. Both rod and cone photoreceptor cells are degenerated with age and all PDE6 subunits degrade in rd10/cpfl1 mice. We expressed cone PDE6α’ in both rods and cones of rd10/cpfl1 mice by adeno-associated virus (AAV)-mediated delivery driven by the ubiquitous, constitutive small chicken β-actin promoter. We show that expression of PDE6α’ rescues rod function in rd10/cpfl1 mice, and the restoration of rod light sensitivity is attained through restoration of endogenous rod PDE6γ and formation of a functional PDE6α’γ complex. However, improved photopic cone responses were achieved only after supplementation of both cone PDE6α’ and PDE6γ’ subunits but not by PDE6α’ treatment alone. We observed a two fold increase of PDE6α’ levels in the eyes injected with both PDE6α’ plus PDE6γ’ relative to eyes receiving PDE6α’ alone. Despite the presence of both PDE6γ’ and PDE6γ, the majority of PDE6α’ formed functional complexes with PDE6γ’, suggesting that PDE6α’ has a higher association affinity for PDE6γ’ than for PDE6γ. These results suggest that the presence of PDE6γ’ augments cone PDE6 assembly and enhances its stability. Our finding has important implication for gene therapy of PDE6α’-associated achromatopsia