JP2017025008A - Treatment of retinitis pigmentosa - Google Patents

Abstract

Disclosed is an adeno-associated virus (AAV) vector used for the treatment of retinitis pigmentosa (RP), which is a phenotype of hereditary retinal dystrophy, for gene therapy of eye diseases. A method of reducing photoreceptor cell death in a subject suffering from retinitis pigmentosa or at risk of developing retinitis pigmentosa, comprising: ciliary neurotrophic factor Administering an adeno-associated virus (AAV) vector containing a nucleotide sequence encoding (CNTF) to a subject in need thereof, wherein visual function is substantially restored in the treated eye, or Maintained, the way. The method wherein the AAV vector contains an AAV serotype 2 genome and the CNTF is human CNTF. A method wherein the AAV vector is administered to the eye of the subject by subretinal injection, direct retinal injection or intravitreal injection. [Selection figure] None

Description

  The present invention relates to a compound used for gene therapy of eye diseases. More particularly, the present invention relates to adeno-associated virus (AAV) vectors for use in the treatment of retinitis pigmentosa (RP), which delivers ciliary neurotrophic factor (CNTF) to the eye. can do.

  Retinitis pigmentosa (RP) is a group of hereditary retinal dystrophy that is linked as a phenotype, resulting in gradual loss of vision. Approximately 1 in 3000-4000 people suffer from RP.

  Early symptoms of RP include reduced night vision and peripheral vision. As the disease progresses, total vision, central vision and color vision may also be affected. The age of onset of RP symptoms varies, but is typically between 10 and 30 years, and the rate of deterioration varies from individual to individual.

  RP is usually caused by progressive degeneration of rod photoreceptors. However, retinal pigment epithelium (RPE) and cone photoreceptors can also degenerate during disease progression.

  Several compounds have been found to have some effect on the maintenance of retinal neurons, including brain-derived neurotrophic factor (BDNF; Okoye, G. et al. (2003) J. Neurosci. 23: 4164-4172), glial cell line-derived neurotrophic factor (GDNF; McGee Sanftner, LH et al. (2001) Mol. Ther. 4: 622-629; Buch, PK et al. (2006) Mol. Ther. 14: 700-709), and ciliary neurotrophic factor (CNTF). CNTF has been shown to prevent retinal ganglion cell death in models of oxidative stress and experimental glaucoma (Ji, JZ et al. (2004) Eur. J. Neurosci. 19: 265-272; Leaver, SG et al. (2006) Eur. J. Neurosci. 24: 3323-3332; MacLaren, RE et al. (2006) Exp. Eye Res. 83: 1118-1127; Maier, K. et al. (2004) Brain 14: 378-387; Pease, ME et al. (2009) Invest. Ophthalmol. Vis. Sci. 50: 2194-2200).

  Numerous studies have demonstrated that sustained expression of CNTF after gene delivery by AAV may result in the protection of photoreceptors for several months in a mouse RP model (Bok, D. et al (2002) Exp. Eye Res. 74: 719-735; Schlichtenbrede, FC et al. (2003) Gene Ther. 10: 523-527; Liang, FQ et al. (2001) Mol. Ther. 3: 241- 248). However, due to the slow progression characteristics of neurodegenerative diseases such as RP, and the short-term nature of these studies, whether observed photoreceptor protection is maintained throughout the life of the animal and is critical Notably, it was previously unknown whether protected neurons retain function.

  Moreover, although these studies observed that CNTF treatment resulted in anatomical preservation of photoreceptors, it was concluded that delivery of CNTF by AAV was not appropriate for the clinical use of RP treatment. Specifically, experiments performed in these studies (eg, electroretinography, ERG) have shown reduced visual function after vector administration (Schlichtenbrede, FC et al. (2003) Gene Ther. 10 : 523-527).

  Another prior approach to delivering CNTF to the eye used encapsulated cell technology, which uses a device that includes cells that produce CNTF. Research on this approach has shown some success in protecting photoreceptors in the medium term, but the applied dose was too low and timely to measure effectiveness in protecting visual function (Birch, DG et al. Long-term follow-up of patients with retinitis pigmentosa (RP) receiving sustained-release CNTF through intraocular encapsulated cell technology implants. ARVO annual meeting (Seattle, WA, 2013); Birch, DG et al. (2013) Am. J. Ophthalmol. 156: 283-292 e281). Furthermore, this technique requires significant surgical intervention to implant the device containing the cells into the subject's eye, and the implant itself can cause safety and immunogenicity problems.

  Currently, no treatment has been found to be good for preventing the onset of RP or improving vision after the onset of illness. Thus, there is still a great need for the treatment of RP, more particularly to prevent the loss of visual function or to enable the visual enhancement of affected individuals.

Okoye, G. et al. (2003) J. Neurosci. 23: 4164-4172 McGee Sanftner, L.H. et al. (2001) Mol. Ther. 4: 622-629 Buch, P.K. et al. (2006) Mol. Ther. 14: 700-709 Ji, J.Z. et al. (2004) Eur. J. Neurosci. 19: 265-272 Leaver, S.G. et al. (2006) Eur. J. Neurosci. 24: 3323-3332 MacLaren, R.E. et al. (2006) Exp. Eye Res. 83: 1118-1127 Maier, K. et al. (2004) Brain Pathol. 14: 378-387 Pease, M.E. et al. (2009) Invest. Ophthalmol. Vis. Sci. 50: 2194-2200 Bok, D. et al. (2002) Exp. Eye Res. 74: 719-735 Schlichtenbrede, F.C. et al. (2003) Gene Ther. 10: 523-527 Liang, F.Q. et al. (2001) Mol. Ther. 3: 241-248 Schlichtenbrede, F.C. et al. (2003) Gene Ther. 10: 523-527 Birch, D.G. et al. Long-term follow-up of patients with retinitis pigmentosa (RP) receiving sustained-release CNTF through intraocular encapsulated cell technology implants.ARVO annual meeting (Seattle, WA, 2013) Birch, D.G. et al. (2013) Am. J. Ophthalmol. 156: 283-292 e281

  The inventors have shown that AAV-mediated CNTF expression provides long-term protection from photoreceptor degeneration in a mouse model. We used repeated retinal imaging to quantify the survival of autofluorescent cone photoreceptors in vivo. Using this new approach, a specific dose range of CNTF was identified that was high enough to stop photoreceptor degeneration without side effects on visual function.

  Surprisingly, the inventors have also demonstrated that living pyramidal cells retain function as well as signals to the brain correctly. Specifically, we used visual cortex imaging and visual evoked behavioral response to evaluate the unexpected beneficial effects of AAV-mediated delivery of CNTF to the eye on visual function. discovered.

  The inventors have given various doses to protect the cone photoreceptors once the rod degeneration has already been ensured, most similar to the eye phenotype of many RP patients visiting the clinic. went. The present inventors combined the standard electrophysiological functional assessment with visual guidance behavioral experiments to measure the degree of functional visual acuity retained after long-term CNTF treatment, while the findings and in the visual cortex Correlates with evidence of advanced signal processing.

  In summary, the present inventors have unexpectedly found that delivery of CNTF to the eye via AAV can be clinically applied to maintenance and treatment of the visual function of RP.

  Thus, in certain embodiments, the present invention provides a method of treating or preventing retinitis pigmentosa, which method comprises adeno-associated virus (N) containing a nucleotide sequence encoding ciliary neurotrophic factor (CNTF). Administration of the AAV) vector to a subject in need thereof.

  The AAV vector may be of any serotype (eg, including any AAV serotype genome and / or capsid protein) as long as the vector has the ability to infect or transduce eye cells. May be).

  In certain embodiments, the AAV vector contains an AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 genome. In another embodiment, the AAV vector contains an AAV serotype 2, 4, 5, or 8 genome. Preferably, the AAV vector contains an AAV serotype 2 genome.

  Preferably, the AAV vector takes the form of an AAV vector particle.

  In certain embodiments, the AAV vector particles contain an AAV2 genome and an AAV2 capsid protein (AAV2 / 2); an AAV2 genome and an AAV5 capsid protein (AAV2 / 5); or an AAV2 genome and an AAV8 capsid protein (AAV2 / 8). Preferably, the AAV vector particle contains an AAV2 genome and an AAV2 capsid protein (AAV2 / 2).

  The AAV vector particles of the present invention may be chimeric, shuffled, or capsid modified derivatives of one or more naturally occurring AAV. Specifically, AAV vector particles can contain capsid protein sequences from different AAV serotypes, clades, clones, or isolates in the same vector (ie, a pseudotype vector). Thus, in certain embodiments, the AAV vector takes the form of a pseudotyped AAV vector particle.

  In certain embodiments, the CNTF is human CNTF.

In another embodiment, the nucleotide sequence encoding CNTF is
(a) a nucleotide sequence having at least 70% identity to SEQ ID NO: 1; and
(b) a nucleotide sequence encoding an amino acid sequence having at least 70% identity to SEQ ID NO: 2,
Selected from the group consisting of
The protein encoded by the nucleotide sequence substantially retains the natural function of the protein represented by SEQ ID NO: 2.

  In another embodiment, the nucleotide sequence encoding CNTF is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. The protein encoded by the nucleotide sequence substantially retains the natural function of the protein represented by SEQ ID NO: 2.

  In another embodiment, the nucleotide sequence encoding CNTF is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. Although it contains a nucleotide sequence that encodes an amino acid sequence having sex, the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 2.

  In certain embodiments, the AAV vector contains a secretory signal sequence operably linked to a nucleotide sequence encoding CNTF. Preferably, the secretion signal sequence is a human nerve growth factor (NGF) secretion signal sequence.

  In certain embodiments, the secretory signal sequence has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3 or 7. A peptide containing a nucleotide sequence having, but encoded by that nucleotide sequence, causes secretion of CNTF operably linked from the expressing cell.

  In certain embodiments, the AAV vector is administered to the eye of the subject by subretinal injection, direct retinal injection, or intravitreal injection. Preferably, the AAV vector is administered to the subject's eye by subretinal injection. Subretinal injection can be performed by the two-stage subretinal injection method described herein.

  In certain embodiments, the AAV vector is administered to the subject in a single dose.

The AAV vector can be, for example, a suspension at a concentration of about 1-2 × 10 ¹¹ , 1-2 × 10 ¹² or 1-2 × 10 ¹³ genomic particles (gp) per mL. Thus, administration of about 2 × 10 ¹⁰ gp AAV vector can be administered, for example, by injecting an approximately 10 μL dose of AAV vector at a concentration of 2 × 10 ¹² gp / mL. One skilled in the art can easily adjust the dosage, volume, and concentration of the AAV vector as needed.

  The volume of AAV vector administered is for example about 1-500 μL, for example about 10-500, 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100-250, 200 -250, 50-150, 1-100 or 1-10 μL. Preferably, the volume of AAV vector composition to be injected is about 100 μL.

In certain embodiments, the AAV vector is administered at a dose of at least 2 × 10 ⁹ , 2 × 10 ¹⁰ , 2 × 10 ¹¹ or 2 × 10 ¹² gp per eyeball. In another embodiment, the AAV vector is administered at a dose of about 2 × 10 ⁹ , 2 × 10 ¹⁰ , 2 × 10 ¹¹ or 2 × 10 ¹² gp per eyeball. Preferably, the AAV vector is administered at a dose of 2 × 10 ¹¹ gp per eye, preferably by subretinal injection.

  The inventors have surprisingly found that even if most or all of the rod cells have degenerated, the retina using the AAV vector of the present invention, for example, by protecting living cone cells. It has been found that the treatment of pigment degeneration may have an effect (eg in maintaining visual function). Accordingly, in certain embodiments, the treated eye has about 100 million, 20 million, 20 million, 10 million, 9 million, 8 million, 7 million, 6 million at the time of administration of the AAV vector. Contains less than 10 million, 4 million, 3 million, 2 million, million, 500000, 250,000, 100,000, or 50,000 rod cells. In another embodiment, the subject is substantially devoid of rod cells in the treated eye at the time of AAV vector administration. In another embodiment, the eye to be treated does not contain rod cells at the time of AAV vector administration.

  In certain embodiments, photoreceptor degeneration due to retinitis pigmentosa is substantially prevented throughout the life of the subject. The photoreceptor cells may include cone cells and / or rod cells, preferably cones and rod cells. In another embodiment, at the time of administration of the AAV vector, about 90%, 80%, 70%, 60%, 50% of the number of photoreceptor cells (eg, pyramidal cells and / or rod cells) present in the eye being treated. Less than%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% are retinal pigment degeneration It then degenerates throughout the life of the subject due to the disease.

  In another embodiment, cone cell degeneration due to retinitis pigmentosa is substantially prevented throughout the life of the subject. In another embodiment, about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15% of the number of pyramidal cells present in the eye being treated at the time of AAV vector administration Less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% will subsequently degenerate throughout the life of the subject due to retinitis pigmentosa . It is preferred that viable cells remain functional.

  In another aspect, the present invention provides a method of reducing photoreceptor cell death in a subject suffering from or at risk of developing retinitis pigmentosa, the method comprising: Administering an adeno-associated virus (AAV) vector containing a nucleotide sequence encoding ciliary neurotrophic factor (CNTF) to a subject in need thereof.

  AAV vectors, CNTF, and administration methods (eg, techniques and dosages) can be as described herein.

  In certain embodiments, the visual function of the treated eye is substantially restored or maintained. Visual function (eg, as measured by the visual function tests described herein) can be restored to, for example, about the same level that was present in the affected eye prior to the onset of retinitis pigmentosa. Alternatively, visual function can be maintained at about the same level in, for example, healthy individuals who may develop retinitis pigmentosa or in subjects who already have retinitis pigmentosa (eg, the present invention). After administration of the AAV vector, there is substantially no loss of visual function or even more as a result of retinitis pigmentosa).

  If left untreated, most or all of the rod cells can degenerate (eg, die) over time as a result of retinitis pigmentosa. Cone cells can also degenerate as the disease progresses.

  In certain embodiments, photoreceptor degeneration due to retinitis pigmentosa is substantially prevented throughout the life of the subject. The photoreceptor cells may include cone cells and / or rod cells, preferably cones and rod cells. In another embodiment, at the time of administration of the AAV vector, about 90%, 80%, 70%, 60%, 50% of the number of photoreceptor cells (eg, pyramidal cells and / or rod cells) present in the eye being treated. Less than%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% are retinal pigment degeneration It then degenerates throughout the life of the subject due to the disease.

  In another embodiment, cone cell degeneration due to retinitis pigmentosa is substantially prevented throughout the life of the subject. In another embodiment, about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15% of the number of pyramidal cells present in the eye being treated at the time of AAV vector administration Less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% will subsequently degenerate throughout the life of the subject due to retinitis pigmentosa . It is preferred that viable cells remain functional.

  In certain embodiments, the treated eye has about 100 million, 50 million, 20 million, 10 million, 9 million, 8 million, 7 million, 6 million, at the time of administration of the AAV vector. Contains less than 5 million, 4 million, 3 million, 2 million, million, 500000, 250,000, 100,000, or 50,000 rod cells. In another embodiment, the subject is substantially devoid of rod cells in the treated eye at the time of AAV vector administration. In another embodiment, the eye to be treated does not contain rod cells at the time of AAV vector administration.

  In another embodiment, the present invention provides an adeno-associated virus (AAV) vector for use in the treatment or prevention of retinitis pigmentosa, wherein the AAV vector comprises ciliary neurotrophic factor (CNTF). Contains the encoding nucleotide sequence.

  The AAV vector, CNTF, method of administration (eg, procedure and dosage) and effect, and subject to be treated can be as described herein.

  In another aspect, the present invention relates to an adeno-associated virus (AAV) vector for use in reducing photoreceptor cell death in a subject suffering from, or at risk of developing retinitis pigmentosa. The AAV vector contains a nucleotide sequence encoding ciliary neurotrophic factor (CNTF).

  The AAV vector, CNTF, method of administration (eg, procedure and dosage) and effect, and subject to be treated can be as described herein.

  In another embodiment, the present invention provides a genetically modified animal for analyzing pyramidal cell survival and / or function, wherein the animal is genetically engineered to contain a detectable marker within the pyramidal cell. Has been modified.

  In certain embodiments, the detectable marker is a fluorescent protein, such as green fluorescent protein.

  In another embodiment, the animal is a mammal, preferably a mouse.

  In another embodiment, the animal is also a disease model animal. Preferably, the animal has been genetically modified to provide a model for retinitis pigmentosa, but the animal is, for example, homozygous for rhodopsin knockout.

(a) Autofluorescence (AF) image of the Rho ^{− / − TgOPN1LW-EGFP +/−} mouse fundus showing cone photoreceptors represented as dotted white dots. (a ′) An enlarged view of AF images of a characteristic group of cone photoreceptors (lined from β to γ) and individual cones (white arrows) at the nasal vessel bifurcation (α, dotted line). (b) Near-infrared (NIR) fundus image showing the retinal vasculature in detail, and (b ′) enlarged NIR image showing the nasal vessel bifurcation (α, dotted line) in detail. (c) Flat mount postmortem retina with retinal vasculature counterstained with lectin. (c ′) An enlarged image of the retinal flat mount showing the distribution of cone photoreceptors (green dots) at the nasal bifurcation (α, dotted line). It should be noted that the cone pattern in panel (c ′) accurately correlates with the pattern observed with in vivo AF imaging (a ′); a characteristic group of cone photoreceptors (from β (lined to γ) and individual cones (white arrows). (dg) Repeated AF fundus imaging of Rho ^{− / − TgOPN1LW-EGFP +/−} mice treated with high doses of rAAV2 / 2.hCNTF from postnatal week 8 (PW) to PW30. (d'-g ') AF enlarged image showing retinal blood vessel life of each animal. Since cone photoreceptors (white dots) can be quantified within the ROI (overlapping red dots), a highly reproducible long-term evaluation of cone survival is possible. Survival is expressed as a function (%) of the number of cone photoreceptors at baseline (PW8). All scale bars are ~ 100 μm. (h) Each treatment group: function of cone photoreceptor number at baseline (PW8) for high dose (dark blue), medium dose (light blue), low dose (light blue), or PBS sham treatment (gray) ( Survival of cone photoreceptors expressed as%). hCNTF treatment significantly affects pyramidal photoreceptor survival: * = p <0.05; ** = p <0.01; *** = p <0.001, repeated measures, using dose and time as factors Original configuration analysis of variance. In PW30, high dose group n = 5, medium dose group n = 6, low dose group n = 8. Near infrared spectroscopy (NIR) and autofluorescence (AF) fundus imaging of high dose rAAV2 / 2.hCNTF treated mice and PBS sham treated mice in PW8 and PW30. Sham-treated controls demonstrate areas of increased reflectivity that show retinal pigment epithelium (RPE) atrophy (a, c) and loss of EGFP-expressing cone photoreceptors (b, d) by PW30. Eyes treated with high doses of rAAV2 / 2.hCNTF show no signs of RPE atrophy (e, g) and minimal reduction in cone photoreceptors (f, h) until the end of the same period . A representative histological image (i) of the retina subjected to PBS sham treatment shows that at the time of PW30, neither rod photoreceptors nor cone photoreceptors are present. The retinal histology (jo) treated with high-dose rAAV2 / 2.hCNTF is a cone (GFP-expressing) and rod (GFP non-expressing) photoreceptor in the outer granular layer (ONL) at PW30. Indicates saving. (Jk) Cone photoreceptor cells (lo) express Mws-opsin in the correct cell compartment (outer segment; OS) (arrowhead) and evidence that opsin is mislocalized in the cell body There is no (*). The cone photoreceptor morphology suggests compression between the inner granular layer (INL) and RPE due to the absence of multiple rows of nuclei after rod degeneration. (Ab) Changes in cortical blood flow (CBF) in ^{PW6 naïve} Rho ^{− / − TgOPN1LW-EGFP +/−} mice, after laser speckle imaging (LSI) stimulation with 510 nm flicker stimulation, It is mainly used to detect small blood flow changes in the visual cortex. (C) Schematic view of the visual pathway in a mouse, indicating that visual input is processed in the visual cortex in the hemisphere opposite the stimulated eye. White arrow = superior sagittal sinus; arrowhead = lambda; OS = left eye (left); OD = right eye (right). PBS treatment (d, g) or rAAV2 / 2.hCNTF (e, h) in PW30 Rho ^{-/-TgOPN1LW-EGFP +/-} mice treated with (de) medium dose and (gi) high dose Changes in CBF were examined after independent eye stimulation and revealed a significant difference in CBF from high-dose treated eyes compared to sham-treated controls (i). ns = not significant; * = p <0.05, Wilcoxon matched-paired signed rank test. LSI: high dose n = 3; medium dose n = 6. (A) Schematic diagram of the optomotor response (OMR) regarding the direction of drum rotation. (B) Number of head tracking responses obtained from individual mice with PW30 (3 tests per animal). Error = SEM; X = no response; L = low dose, M = medium dose, H = high dose. (C) Group average of responses to OMR tested with PW30, where the number of headtracks obtained from the medium dose (light blue) and high dose (dark blue) was higher than the paired PBS sham-treated controls Significantly more. ns = not significant, *** = p <0.001, one-way analysis of variance. Low dose n = 5, medium dose n = 6, high dose n = 4. (A) Hierarchical clustering of transcriptome output from low-dose, medium-dose, and high-dose rAAV2 / 2.hCNTF-treated mice, which were paired with PBS sham-treated controls (in group 1) The change in gene expression for 4 mice) is shown, and the blue solid line in each column represents the fold change relative to the baseline (dotted line). Expanding the two clusters for genes with the largest transcriptional changes: upper diagram shows gene down-regulation for ion transport and visual cycle; lower diagram for genes encoding serine-, cysteine-, and metallopeptidase inhibitors Shows significant up-regulation. (B) Gene clustering based on functions using the concept of Genome Search Meta Analysis (GMSA), which was visualized using the Enrichment Map plug-in for Cytoscape. Detection of hCNTF protein levels by ELISA from (a) culture medium aspirated from HEK293 cells after transduction with rAAV2 / 2.hCNTF or (b) eyes injected with rAAV2 / 2.hCNTF , The potent secretory effect of hCNTF, and the possibility of roughly controlling the dose by changing the number of administered vector particles. (Cj) Repeated AF fundus imaging of Rho ^{− / − TgOPN1LW-EGFP +/−} mice treated with high doses of rAAV2 / 2.hCNTF from PW8 to PW30. (C'-j ') AF magnified image showing how retinal vessel markers can be used to determine areas of interest (ROI) that remain constant throughout the life of the animal. Since cone photoreceptors (white dots) can be quantified within the ROI (overlapping red dots), a highly reproducible long-term evaluation of cone survival is possible. Survival is expressed as a function (%) of the number of cone photoreceptors at baseline (PW8). All scale bars are ~ 100 μm. * = p <0.05, Mann-Whitney U test; n = 3 wells / group (a); n = 3 eyes / group (b). Representative near-infrared (NIR) and autofluorescence (AF) fundus imaging of Rho ^{-/-TgOPN1LW-EGFP +/-} mice, devoted to each treatment group: (a) high dose; (b) medium dose Or (c) low dose rAAV2 / 2.hCNTF; or (d) PBS sham treatment. The dose is given as genomic particles (gp) in a 2 μL volume administered per eye. Cone photoreceptors are observed in autofluorescence (AF) mode as if they were white dots. RPE atrophy can be observed as an increase in brightness by NIR imaging, but can be observed at later time points, especially in the low dose (c vii-viii) and PBS sham-treated (d vi-viii) groups. Rho ^{-/-TgOPN1LW-EGFP +} treated with one eye (a) low dose (light blue), (c) medium dose (light blue), or (e) high dose rAAV2 / 2.hCNTF (dark blue) // ^- Representative electroretinography (ERG) traces of mice compared to paired PBS sham-treated eyes (gray) (b, d, f). Measurement of light-adapted b-wave in response to a 1 Hz 25 cd.s / m ² flash stimulus (30 cd / m ² multicolor white light background) at PW8, 10, and 12, showing b-wave amplitude . Light-adapted b-wave amplitudes were absent or below recordable levels in all groups up to 12 weeks. * = p <0.05; ** = p <0.01; *** = p <0.001, multiple t-test with Bonferroni post-correction. (a) Correlation between cone photoreceptor survival and OMR score for PW30 individual mice treated with medium and high dose treatment; solid line = linear regression, dotted line = 95% confidence interval. r ² = 0.80, F = 33, p = 0.0004. (b) Graphical display showing transcriptome analysis of eyes that received high dose treatment versus PBS sham-treated controls; 23365 transcripts (grey dots) are log2 vs. normalized mean Differential expression variation based on fold change was evaluated. The 1533 genes (red dots) were found to have significant expression level differences in eyes receiving high dose rAAV2 / 2.hCNTF compared to paired sham-treated controls.

Explanation of Table 1 Selected genes are classified by gene ontology. The gene symbol (UCSC, mm9), gene name, fold change, log2 ratio, and significance are shown for each gene. Up-regulated genes = blue shading; down-regulated genes = red shading.

DETAILED DESCRIPTION OF THE INVENTION Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

  The practice of the present invention will utilize conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology, unless otherwise indicated, but these techniques are within the ability of one skilled in the art. It is. Such techniques are explained in the literature. For example, Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley &Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley &Sons; Polak, JM and McGee , J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM and Dahlberg, JE (1992) Methods See in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. These general references are incorporated herein by reference.

In certain embodiments, the present invention provides a method of treating or preventing retinitis pigmentosa, the method comprising adeno-associated virus (AAV) containing a nucleotide sequence encoding ciliary neurotrophic factor (CNTF). Administering the vector to a subject in need thereof.
Retinitis pigmentosa (RP)
Retinitis pigmentosa (RP) is a group of hereditary retinal dystrophy that is related as a phenotype, usually caused by progressive degeneration of rod photoreceptors. Retinal pigment epithelium (RPE) and cone photoreceptors can also degenerate during disease progression.

  RP is characterized by changes in retinal pigment as a clinical finding, which may be accompanied by arteriolar narrowing and optic nerve atrophy. Retinal changes may be due to retinal pigment dispersion and aggregation. This can give rise to a wide range of appearances ranging from granular or speckled to bone-like specific local aggregates. A black or dark brown star-shaped group of pigments may appear. In addition, pigmentation confined to the quadrants of the retina, abnormalities that appear to radiate from the disc, and changes with severe vascular damage may be observed.

  Treatment or prevention of RP as described herein can reduce or prevent the appearance of the RP phenotype described above. It can have the consequence of protecting the photoreceptor cells, eg cone cells, from degeneration. Preferably, the treatment protects both cones and rod cells from degeneration.

  One skilled in the art can estimate the number of rods and cones at the clinic using techniques such as adaptive optics, autofluorescence, and optical coherence tomography (OCT) scanning.

  Treatment of RP preferably allows maintenance or improvement of visual function.

One skilled in the art can easily perform visualization of the appearance of the retina and evaluation of visual function. For example, visual function tests performed by those skilled in the art include maximum corrected visual acuity, visual field test, microperimetry, color vision, dark adaptation test, electroretinogram test, and cone flicker value measurement test. As used herein, “maintaining or improving visual function” refers to one or more of the above when comparing the vision of a treated eye before and after performing the method of the present invention. It is to be understood that the visual as assessed by the visual function test is maintained at substantially the same level or that the visual level is improved.
Ocular Structure The agents described herein can be administered to a mammal, preferably a human eye, in connection with the treatment or prevention of retinitis pigmentosa (RP).

Those skilled in the treatment of eye diseases have a detailed and complete understanding of the structure of the eye. However, the following structures that are particularly relevant to the present invention will be described.
The retina retina is a multi-layered membrane that covers the internal posterior chamber of the eye, senses the image of the visual world, and transmits the image to the brain through nerves. In order from the inside to the outside of the eye, the retina has a layer of retinal neurosensory epithelium and retinal pigment epithelium, and there is a choroid outside the retinal pigment epithelium.
Retinal neurosensory epithelium and photoreceptors The retinal neurosensory epithelium has photoreceptors in it that directly sense light. This includes the following layers: inner boundary membrane (ILM); nerve fiber layer; ganglion cell layer; inner plexiform layer; inner granule layer; outer reticular layer; outer granule layer (photonuclear); It comprises photoreceptor cells (inner and outer segments) consisting of rods and cones.

  Those skilled in the art have a detailed understanding of photoreceptor cells. In short, photoreceptor cells are specialized neurons in the retina that convert light into biological signals. Visual cells include rods and cones, which are distributed differently across the retina.

  Rod cells are mainly distributed in the outer part of the retina. These cells are sensitive and provide vision with low light. The normal human retina has an average of about 125 million rod cells.

Cone cells are found throughout the retina, but are present at a particularly high density in the fovea, which is a depression of the retinal neurosensory epithelium, and this fovea carries high-resolution central vision. The sensitivity of cone cells is lower than that of rod cells. The normal human retina has an average of about 6-7 million cone cells.
Retinal pigment epithelium The retinal pigment epithelium (RPE) is a pigmented cell layer just outside the retinal neurosensory epithelium. RPE performs many functions, including the transport of nutrients and other substances to photoreceptor cells, and the absorption of scattered light to improve vision.
The choroidal choroid is a vascular rich layer between the sclera outside the eye and the RPE. The choroidal vasculature can supply the retina with oxygen and nutrients.
Ciliary neurotrophic factor (CNTF)
Ciliary neurotrophic factor (CNTF) is a polypeptide hormone and nerve growth factor that has been shown to be a survival factor for neurons and oligodendrocytes. In addition, CNTF has been found to promote neurotransmitter synthesis and neurite outgrowth in specific neuronal populations.

  In certain embodiments of the invention, the CNTF is human CNTF.

  In one embodiment, the nucleotide sequence encoding CNTF is the sequence registered under NCBI accession number NM_000614.

（配列番号1）
ある実施形態において、CNTFのアミノ酸配列は、NCBIアクセッション番号NP_000605とされる配列である。 In another embodiment, the nucleotide sequence encoding CNTF is:

(SEQ ID NO: 1)
In one embodiment, the amino acid sequence of CNTF is the sequence designated NCBI accession number NP_000605.

（配列番号2）
本発明のCNTFをコードするヌクレオチド配列はたとえば、配列番号1に対して少なくとも70%、80%、90%、95%、96%、97%、98%、99%、または100%の同一性を有するヌクレオチド配列を含んでいてもよく、そのヌクレオチド配列によりコードされるタンパク質は、配列番号2で表されるタンパク質の天然の機能を実質的に保持している。 In another embodiment, the amino acid sequence of CNTF is:

(SEQ ID NO: 2)
The nucleotide sequence encoding CNTF of the present invention has, for example, at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. The protein encoded by the nucleotide sequence substantially retains the natural function of the protein represented by SEQ ID NO: 2.

  The nucleotide sequence encoding CNTF of the present invention has, for example, at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2. And the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 2.

Preferably, the nucleotide sequences of the present invention in a subject suffering from or at risk of developing RP,
(a) Clinical findings of RP-related retinal pigment changes
(b) death of photoreceptor cells (eg, cone cells, preferably cone and rod cells); and / or
(c) Encodes a protein that helps prevent or reduce visual function loss as much or better than the protein of SEQ ID NO: 2.
Secretion signal sequence In one embodiment of the invention, the AAV vector contains a secretion signal sequence operably linked to a nucleotide sequence encoding CNTF.

  A signal sequence is generally a short peptide at the N-terminus of a protein that directs the protein into the cell's secretory pathway. As used herein, a “secretory signal sequence” is a peptide that facilitates secretion of a protein (eg, CNTF) from a cell, or a nucleotide sequence that encodes the peptide, as necessary.

  The peptide of the secretory signal sequence is cleaved from the protein when it is secreted from the cell, resulting in the mature form of the protein.

  In certain embodiments, the secretion signal sequence is a human neurotrophic factor (NGF) secretion signal sequence.

  In another embodiment, the secretory signal sequence is the signal sequence of the NGF nucleotide sequence designated NCBI accession number NM_002506 (eg, the sequence corresponding to nucleotides 170-223 of the mRNA numbered). .

（配列番号3）
もう1つの実施形態において、分泌シグナル配列は下記である：

（配列番号7）
本発明の分泌シグナル配列は、たとえば、配列番号3または7に対して少なくとも70%、80%、90%、95%、96%、97%、98%、99%、または100%の同一性を有するヌクレオチド配列を含んでいてもよく、そのヌクレオチド配列によってコードされるペプチドが、機能しうるように連結されているCNTFを、その発現細胞から分泌させる。 In another embodiment, the secretory signal sequence is:

(SEQ ID NO: 3)
In another embodiment, the secretory signal sequence is:

(SEQ ID NO: 7)
The secretory signal sequence of the invention has, for example, at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3 or 7. It may contain a nucleotide sequence having, and the CNTF operably linked to the peptide encoded by the nucleotide sequence is secreted from the expression cell.

  “Operatively linked” is understood to mean that the individual components are joined together so that they can perform their function without substantial interference (eg, secretion). The signal sequence is operably linked to the nucleotide of interest and can facilitate secretion of the protein encoded by the nucleotide of interest from the cell; alternatively, the promoter can function to the nucleotide of interest. To promote expression of the nucleotide of interest in the cell).

（配列番号8） - ヒトNGF分泌シグナル配列に下線を付す

（配列番号9） - ヒトNGF分泌シグナル配列に下線を付す
本発明のある実施形態において、AAVベクターは、配列番号8に対して少なくとも70%、80%、90%、95%、96%、97%、98%、99%、または100%の同一性を有するヌクレオチド配列を含有し、そのヌクレオチド配列によりコードされるタンパク質は、配列番号9で表されるタンパク質の天然の機能を実質的に保持する。 SEQ ID NOs: 8 and 9 correspond to an example of a nucleotide sequence and an amino acid sequence, respectively, of a human NGF secretion signal sequence operably linked to a human CNTF sequence.

(SEQ ID NO: 8)-human NGF secretion signal sequence is underlined

(SEQ ID NO: 9)-human NGF secretion signal sequence is underlined In certain embodiments of the invention, the AAV vector is at least 70%, 80%, 90%, 95%, 96%, 97 relative to SEQ ID NO: 8. A protein that contains a nucleotide sequence having%, 98%, 99%, or 100% identity and that is encoded by that nucleotide sequence substantially retains the natural function of the protein represented by SEQ ID NO: 9 .

In another embodiment of the invention, the AAV vector has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9. The amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 9.
Vector vectors are tools that allow or facilitate the transfer of a subject from one environment to another.

The vector used in the present invention can contain a promoter for expression of the polynucleotide, and if necessary, a promoter regulatory element.
Adeno-associated vector (AAV) vector The vector of the present invention is an adeno-associated virus (AVV) vector. The AAV vector is preferably in the form of AAV vector particles.

  Methods for preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art and can be readily adapted by those skilled in the art for the required purpose.

  AAV vectors can contain the AAV genome or a derivative thereof.

  The AAV genome is a polynucleotide sequence that encodes functions necessary for the production of AAV particles. Such functions may operate in the replication and packaging of AAV in the host cell, including encapsidating the AAV genome into capsids into AAV particles. Naturally occurring AAV is replication deficient and will rely on providing helper functions in trans to complete the replication and packaging cycle. Thus, the AAV genome of the vectors of the present invention is typically replication defective.

  The AAV genome is single-stranded and may be positive or negative, but can also be double-stranded. The use of the double stranded form can allow the bypass of the DNA replication step in the target cell and promote transgene expression.

  The AAV genome is obtained from any naturally occurring serotype, isolate, or clade of AAV. Thus, the AAV genome can be said to be a complete genome of naturally occurring AAV. As known to those skilled in the art, naturally occurring AAV can be classified according to various biological systems.

  AAV is usually mentioned in terms of its serotype. The serotype corresponds to different variants of AAV, but it has unique reactivity due to its capsid surface antigen expression characteristics, which can be used to remove it from various other variants. Can be distinguished. Typically, viruses with a particular AAV serotype do not efficiently cross-react with neutralizing antibodies specific for any other AAV serotype.

  AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, and recombinant serotypes such as Rec2 and Rec3, which have recently been identified from primate brain It was. Any of these AAV serotypes can be used in the present invention. Thus, in certain embodiments of the invention, the AAV vector particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rec2, or Rec3 AAV vector particles.

  A preferred AAV serotype for use in the present invention is AAV2.

  Other serotypes of particular interest for use in the present invention include AAV4, AAV5, and AAV8, which efficiently transduce eye tissues such as the retinal pigment epithelium. .

  A review on AAV serotypes can be found in Choi et al. (2005) Curr. Gene Ther. 5: 299-310 and Wu et al. (2006) Molecular Therapy 14: 316-27. The sequence of the AAV vector genome used in the present invention, or the sequence of the elements of the AAV genome including the ITR sequence, rep, cap gene, etc. is obtained from the following accession number for the entire AAV genome sequence: Adeno-associated virus 1 NC_002077, AF063497; Adeno-associated virus 2 NC_001401; Adeno-associated virus 3 NC_001729; Adeno-associated virus 3B NC_001863; Adeno-associated virus 4 NC_001829; Adeno-associated virus 5 Y18065, AF085716; Adeno-associated virus 6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-1 NC_006263, AY629583; Bovine AAV NC_005889, AY388617.

  AAV may also be mentioned in terms of clades or clones. This relates to the phylogenetic affinity of naturally occurring AAV and is typically related to the phylogenetic group of AAV, which can be traced back to a common ancestor and includes all of its progeny. In addition, AAV may be referred to with respect to a particular isolate, ie a genetic isolate of a particular naturally occurring AAV. The term genetically isolated population refers to an AAV population that has received little genetic mixing with other naturally occurring AAV, and thus refers to populations that are readily distinguishable at the genetic level.

  One skilled in the art can select an appropriate serotype, clade, clone, or isolate of AAV for use in the present invention based on well-known general knowledge. For example, AAV5 capsid has been shown to transduce primate cone photoreceptors, as evidenced by successful correction of hereditary color blindness (Mancuso et al. (2009) Nature 461: 784- 7).

  The AAV serotype determines the tissue specificity (or affinity) of AAV virus infection. Therefore, preferred AAV serotypes for use in AAV administered to patients in accordance with the present invention are those that have a natural affinity for target cells inside the eye or have a high infection efficiency for those cells. is there. In certain embodiments, the AAV serotype used in the present invention is one that infects retinal neurosensory epithelium, retinal pigment epithelium, and / or choroidal cells.

  Typically, the AAV naturally occurring serotype, isolate, or clade AAV genome contains at least one inverted terminal repeat (ITR). The ITR sequence acts on cis to provide a functional origin of replication and allow vector integration and excision of the vector from the cell genome. In a preferred embodiment, one or more ITR sequences are flanked by nucleotide sequences encoding CNTF. AAV genomes typically also contain packaging genes, such as rep and / or cap genes, which encode the packaging functions for AAV particles. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52, and Rep40, or variants thereof. The cap gene encodes one or more capsid proteins, such as VP1, VP2, and VP3, or variants thereof. These proteins constitute the capsid of AAV particles. Capsid variants are discussed below.

  A promoter is operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19, and p40 promoters (Laughlin et al. (1979) Proc. Natl. Acad. Sci. USA 76: 5567-5571). For example, the p5 and p19 promoters are commonly used for rep gene expression, whereas the p40 promoter is generally used for cap gene expression.

  Therefore, as described above, the AAV genome used in the vector of the present invention can be the whole genome of AAV that exists in nature. For example, AAV vectors or vector particles can be prepared in vitro using vectors containing the entire AAV genome. However, although it is possible in principle to administer such a vector to a patient, it is rarely done in practice. Preferably, the AAV genome is derivatized for administration to a patient. Such derivatization is common in the art, and the present invention uses the use of any known AAV genomic derivative, as well as derivatives that can be made by applying known techniques in the art. Include. Derivatization of the AAV genome and AAV capsid is reviewed in Coura and Nardi (2007) Virology Journal 4: 99, and Choi et al. And Wu et al., Supra.

  Derivatives of the AAV genome include any truncated or modified form of the AAV genome, which allows the expression of the transgene from the vector of the invention in vivo. In general, but importantly, it is possible to truncate the AAV genome so that it retains the above functions while containing minimal viral sequences. This is a safety measure in order to reduce the risk of recombination between the vector and wild-type virus, and to avoid the induction of a cellular immune response due to the presence of the viral gene protein in the target cell. Preferred for reasons.

  Typically, the derivative contains at least one inverted terminal repeat (ITR), preferably two or more ITRs, such as two ITRs or three or more. One or more ITRs can be obtained from AAV genomes having different serotypes, but may also be chimeric or mutant ITRs. Preferred mutant ITRs have trs (terminal separation site) deletions. This deletion allows for continuous replication of the genome, producing a single-stranded genome that contains both the coding and complementary sequences, ie, a self-complementary AAV genome. This allows for the promotion of transgene expression as it results in DNA replication bypass in target cells.

  One or more ITRs are preferably flanked by nucleotide sequences encoding CNTF at either end. Containing one or more ITRs means, for example, that a single-stranded vector DNA is converted into double-stranded DNA by the action of host cell DNA polymerase, and then the concatamers of the vector of the present invention are formed in the nucleus of the host cell. Preferred for promoting. The formation of such episomal concatamers protects the vector construct during the life of the host cell, thereby allowing long-term expression of the transgene in vivo.

  In a preferred embodiment, the ITR element will be the only sequence retained in the derivative from the native AAV genome. Accordingly, it is preferred that the derivative does not contain the rep and / or cap gene of the natural genome and any other sequence of the natural genome. This is also preferred for the reasons described above as well as reducing the possibility of integration of the vector into the host cell genome. In addition, reducing the size of the AAV genome can increase flexibility in incorporating other sequence elements (eg, regulatory elements) into the vector in addition to the transgene.

  Thus, in the derivatives of the present invention, the following parts can be removed: one inverted terminal repeat (ITR) sequence, a replication (rep) and a capsid (cap) gene. However, in certain embodiments, the derivative can additionally contain one or more rep and / or cap genes, or other viral sequences of the AAV genome. Naturally occurring AAV is frequently integrated into specific sites on human chromosome 19 and exhibits negligible frequency of random integration, but retention of the vector's integration ability can be tolerated in the therapeutic setting.

  Where the derivative contains a capsid protein, ie VP1, VP2, and / or VP3, the derivative can be one or more naturally occurring chimeric derivatives, shuffle derivatives, or capsid modified derivatives of AAV. More particularly, the present invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates of AAV in the same vector (ie, a pseudotype vector).

  Chimeric, shuffled, or capsid modified derivatives are typically selected to provide the viral vector with one or more desired functionalities. Therefore, these derivatives have increased gene transfer efficiency, reduced immunogenicity (humoral or cellular), viral affinity range compared to AAV vectors containing naturally occurring AAV genomes such as AAV2. It may show changes and / or improved targeting to specific cell types. Increases in gene transfer efficiency include increased receptor or co-receptor binding at the cell surface, improved internalization, improved transport into cells and nuclei, improved virulence of virus particles, and doubles from single-stranded genomes May be achieved by improved conversion to chain form. Since increased efficiency may also be related to changes in the affinity region of the virus, or targeting to specific cell populations, the vector is not diluted by administration to unwanted tissue.

  Chimeric capsid proteins include those produced by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This can be done, for example, by a marker rescue approach, in which a non-infectious capsid sequence of one serotype is cotransfected with a capsid sequence of another serotype and the desired properties are determined by the specified selection. A capsid sequence having is selected. Different serotype capsid sequences can be altered in cells by homologous recombination to create new chimeric capsid proteins.

  Chimeric capsid proteins move specific capsid protein domains, surface loops, or specific amino acid residues between two or more capsid proteins, for example, between two or more capsid proteins of different serotypes Thus, those produced by manipulating capsid protein sequences are also included.

  Shuffle or chimeric capsid proteins can also be made by DNA shuffling or error-prone PCR. Hybrid AAV capsid genes are generated by randomly fragmenting sequences of related AVV genes, such as sequences encoding capsid proteins of different serotypes, and then reconstituting the fragments with a self-priming polymerase reaction. This reaction can also cause crossover in regions of sequence homology. Thus, a library of hybrid AAV genes generated by shuffling several serotype capsid genes can be screened to identify viral clones with the desired functionality. Similarly, error-prone PCR can be used to generate a library of diverse variants that can be randomly mutated in the AAV capsid gene and then selected for desirable properties.

  Capsid gene sequences can also be genetically modified to introduce specific deletions, substitutions, or insertions relative to the native wild-type sequence. Specifically, the capsid gene can be modified by inserting an irrelevant protein or peptide sequence within the open reading frame of the capsid coding sequence, or at the N and / or C terminus of the capsid coding sequence.

  An irrelevant protein or peptide is advantageous if it functions as a ligand for a particular cell type, thereby providing improved binding to the target cell or increasing the targeting specificity of the vector for a particular cell population. be able to. An example is the use of RGD peptides, which can block uptake in the retinal pigment epithelium and thereby promote the transduction of surrounding retinal tissue (Cronin et al. (2008) ARVO Abstract: D1048) . The irrelevant protein may be one that aids in the purification of the viral particle as part of the manufacturing process, ie an epitope or an affinity tag. The insertion site should typically be selected so as not to interfere with other functions of the viral particle, such as internalization, viral particle transport. A person skilled in the art can identify a site suitable for insertion based on the well-known general knowledge. Individual sites are described in Choi et al., Cited above.

  The present invention further includes the provision of sequences of the AAV genome that have a different order and arrangement from the sequence of the native AAV genome. The invention also includes the replacement of one or more AAV sequences or genes with sequences from another virus, or with chimeric genes composed of sequences from two or more viruses. Such a chimeric gene can consist of sequences derived from two or more related viral proteins of different viral species.

  The vectors of the present invention can take the form of nucleotide sequences containing sequences encoding the AAV genome or derivatives thereof and the CNTF transgene or variants thereof.

  AAV particles of the present invention include capsid-exchanged forms in which an AAV genome or derivative having a certain serotype of ITR is packaged within a different serotype capsid. Some AAV particles of the present invention are mosaic-type, in which a mixture of unmodified capsid proteins derived from two or more different serotypes constitutes the virus particles. Some AAV particles are chemically modified with a ligand adsorbed on the capsid surface. For example, such ligands can include antibodies for targeting specific cell surface receptors.

Thus, for example, the AAV particles of the present invention include those having AAV2 genome and AAV2 capsid protein (AAV2 / 2), those having AAV2 genome and AAV5 capsid protein (AAV2 / 5), and AAV2 genome and AAV8 capsid protein. There are things (AAV2 / 8) etc.
Promoters and Control Sequences The vectors of the present invention may also contain elements that allow the expression of CNTF transgenes in vitro and in vivo. Such elements are sometimes referred to as expression control sequences. Thus, vectors typically contain expression control sequences (eg, including promoter sequences) operably linked to the nucleotide sequence encoding the transgene.

  Any suitable promoter may be used, and those skilled in the art can easily select it. A promoter sequence may function constitutively (ie, operate in any host cell environment), but only operates in a specific host cell environment, thus allowing targeted expression of the transgene in a specific cell type (Eg, tissue specific promoters). A promoter may exhibit inducible expression depending on the presence of another factor, such as a factor present in the host cell. In any case, when the vector is administered for therapy, it is preferred if the promoter functions in the target cell environment.

  In certain embodiments, it is preferred that the promoter exhibits expression specific to retinal cells so as to allow the transgene to be expressed only in the retinal cell population. Thus, expression from the promoter is retinal cell specific and can be limited to cells of the retinal neurosensory epithelium and retinal pigment epithelium, for example.

（配列番号4）
網膜特異的な遺伝子発現を引き起こす、ヒト配列に基づくプロモーターの例としては、桿体および錐体のロドプシンキナーゼ（Allocca et al. (2007) J. Virol. 81: 11372-80）、錐体だけのPR2.1（Mancuso et al. (2009) Nature 461: 784-7）、および/または網膜色素上皮のRPE65（Bainbridge et al. (2008) N. Engl. J. Med. 358: 2231-9）などがある。 A preferred promoter is the chicken β-actin (CBA) promoter, which may be combined with a cytomegalovirus (CMV) enhancer element as appropriate. An example of a promoter used in the present invention is a hybrid CBA / CAG promoter, such as the promoter used in the rAVE expression cassette (GeneDetect.com). Another example promoter used in the present invention has the following sequence:

(SEQ ID NO: 4)
Examples of human sequence-based promoters that cause retina-specific gene expression include rod and cone rhodopsin kinases (Allocca et al. (2007) J. Virol. 81: 11372-80), cone-only PR2.1 (Mancuso et al. (2009) Nature 461: 784-7) and / or retinal pigment epithelium RPE65 (Bainbridge et al. (2008) N. Engl. J. Med. 358: 2231-9) etc. There is.

  The vectors of the present invention may additionally contain one or more regulatory sequences that act before or after transcription. The control sequence may be part of the natural transgene locus, but may also be a heterologous control sequence. The vectors of the present invention can contain a portion of a 5′-UTR or 3′-UTR derived from a natural transgene transcript.

（配列番号5）
本発明のベクターに関して、このような制御配列はシス作用性である。しかしながら、本発明は、追加の遺伝子構築物上にあるトランス作用性制御配列の使用も包含する。 A control sequence is any sequence that facilitates transgene expression, ie, any sequence that acts to increase expression of a transcript, improve mRNA nuclear export, or enhance its stability. Such control sequences include, for example, enhancer elements, post-transcriptional control elements, and polyadenylation sites. A preferred polyadenylation site is the bovine growth hormone poly A signal as shown below:

(SEQ ID NO: 5)
For the vectors of the present invention, such control sequences are cis acting. However, the present invention also encompasses the use of trans-acting regulatory sequences on additional genetic constructs.

（配列番号6）
本発明はまた、WPREなしのベクターと比べて導入遺伝子の発現を増大させるWPREの任意のバリアント配列の使用も包含する。バリアント配列は、配列番号6に対してその配列全体を通して少なくとも70%の相同性を示すことが好ましく、配列番号6に対してその配列全体を通してより好ましくは75%、80%、85%、90%、さらにより好ましくは少なくとも95%、96%、97%、98%、または99%相同性を示す。 A preferred post-transcriptional control element for use in the vectors of the invention is the woodchuck hepatitis virus post-transcriptional control element (WPRE) or a variant thereof. A typical sequence of WPRE is shown below:

(SEQ ID NO: 6)
The invention also encompasses the use of any variant sequence of WPRE that increases transgene expression relative to a vector without WPRE. The variant sequence preferably exhibits at least 70% homology to SEQ ID NO: 6 throughout the sequence, more preferably 75%, 80%, 85%, 90% throughout the sequence to SEQ ID NO: 6. Even more preferably at least 95%, 96%, 97%, 98%, or 99% homology.

Another control sequence that can be used in the vectors of the invention is the scaffold attachment region (SAR). Additional control sequences can also be readily selected by those skilled in the art.
In certain embodiments of the invention, the AAV vector is administered to the eye of the subject by subretinal injection, direct retinal injection, or intravitreal injection.

  Those skilled in the art are familiar with and can perform individual subretinal injections, direct retinal injections, or intravitreal injections.

Preferably, the AAV vector is administered by subretinal injection.
Subretinal injection A subretinal injection is an injection into the subretinal space, ie just below the retinal neurosensory epithelium. During subretinal injection, the injected material is directed to the photoreceptor cells and the retinal pigment epithelium (RPE) layer, creating a space between them.

  If the injection is made by a small retinal incision, retinal detachment can be caused. The exfoliated and raised layer caused by the injected material is called a “bleb”.

  The pores created by subretinal injection must be small enough so that the injected solution does not significantly flow back into the vitreous cavity after administration. Such reflux will be particularly problematic during drug injection because the drug's effect may be diverted from the target zone. The injection preferably creates an injection hole that closes naturally in the retinal neurosensory epithelium, i.e., once the injection needle is removed, the hole made of the needle is unlikely to release the injected material through the hole. Or it is closed again so that there is virtually no.

  To facilitate this process, dedicated subretinal needles are commercially available (eg, DORC 41G Teflon subretinal injection needle, Dutch Ophthalmic Research Center International BV, Zuidland, The Netherlands). These are needles designed for performing subretinal injections.

  As long as there is no damage to the retina at the time of injection and a sufficiently thin needle is used, virtually all of the injected material will be removed from the detached retinal neurosensory epithelium and RPE at the local retinal detachment site. Continue to localize (ie, do not flow back into the vitreous cavity). In fact, typical short-term bleb persistence usually indicates that there is little infusion of the injected material into the vitreous. The bleb will disappear over time as the injected material is absorbed.

Visualization of the eye, particularly the retina, using optical coherence tomography, for example, can be performed preoperatively.
Two-stage subretinal injection The vector of the present invention can be administered with increased accuracy and safety by using a two-stage method in which local retinal detachment is caused by subretinal injection of the first solution. The first solution contains no vector. The second subretinal injection is then used to deliver the vector-containing drug into the bleb's subretinal fluid produced by the first subretinal injection. Since the injection that delivers the drug should not be used to detach the retina, an aliquot of the solution can be injected in the second stage.

In certain embodiments of the invention, the AAV vector is delivered by:
(a) administering to a subject by subretinal injection a sufficient amount of a solution to at least partially detach the retina to form a subretinal bleb, the solution containing no vector;
(b) administering a drug composition by subretinal injection into the bleb formed in step (a), the drug composition containing a vector.

  The amount of solution injected in step (a) to at least partially detach the retina is, for example, about 10-1000 μL, such as about 50-1000, 100-1000, 250-1000, 500-1000, 10 -500, 50-500, 100-500, 250-500 μL. The amount can be, for example, about 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μL.

  The amount of pharmaceutical composition injected in step (b) is, for example, about 10-500 μL, such as about 50-500, 100-500, 200-500, 300-500, 400-500, 50-250, 100- It can be 250, 200-250, or 50-150 μL. The amount can be, for example, about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μL. Preferably, the amount of pharmaceutical composition injected in step (b) is 100 μL. Larger amounts may increase the risk of forcibly extending the retina, but smaller amounts can be difficult to see.

The solution containing no drug (ie, the “first solution” in step (a)) can be prepared in the same manner as the above solution containing the drug substance. A preferred solution that does not contain a drug is a balanced salt solution (BBS) or similar buffer solution adapted to the pH and osmotic pressure of the subretinal space.
Intraoperative visualization of the retina Under certain circumstances, for example, at the end of retinal degeneration, it is difficult to identify the retina, but it is a thin, transparent retina with discolored pigmentation on which the retina rests. This is because it is difficult to see the strong epithelium as a background. The use of blue vital dyes (eg, Brilliant Peel®, Geuder; Membrane Blue-Dual®, Dorc) can be used in retinal detachment procedures (ie, the steps of the two-stage subretinal injection method of the present invention (a The drug can be administered through the same hole without the risk of reflux into the vitreous cavity because it can facilitate the identification of the retinal hole created for)).

The use of blue vital stains also identifies any area of the retina where there is a thickened inner limiting membrane or epiretinal membrane, but injections through any of these tissues can be used to skillfully access the subretinal space Will hinder. Furthermore, contracting any of the tissue structures immediately after surgery can lead to forcibly stretching the injection hole in the retina, which can cause drug backflow into the vitreous cavity.
Pharmaceutical Compositions and Injection Solutions The agents of the present invention, such as vectors, can be formulated into pharmaceutical compositions. Such compositions can contain, in addition to the drug, pharmaceutically acceptable bases, diluents, additives, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials need not be toxic and do not interfere with the effectiveness of the active ingredient. The exact characteristics of the base and other substances can be determined by those skilled in the art depending on the route of administration, eg subretinal injection, direct retinal injection or intravitreal injection.

  The pharmaceutical composition typically takes the form of a liquid. Liquid pharmaceutical compositions generally contain a liquid base such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Saline, magnesium chloride, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included. In some cases, a surfactant such as 0.001% pluronic acid (PF68) may be used.

  For injection into the affected area, the active ingredient can be in the form of an aqueous solution that is free of pyrogens and has a stable pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic solvents such as, for example, sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as needed.

For delayed release, the drug is incorporated into a pharmaceutical composition formulated for sustained release according to methods known in the art, for example, as microcapsules formed from biocompatible polymers or as a liposome delivery system. It may be included.
Methods of Treatment It will be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment; however, in the present invention, reference to prevention is more generally associated with prophylactic treatment. . Treatment can also include stopping the progression of disease severity.

The treatment of mammals, particularly humans, is preferred. However, both human and veterinary treatments are within the scope of the present invention.
Variants, derivatives, analogs, homologs, and fragments In addition to the specific proteins and nucleotides described herein, the invention also encompasses the use of those variants, derivatives, analogs, homologs, and fragments.

  In the context of the present invention, a variant of any given sequence is such that a particular sequence of residues (whether amino acids or nucleic acids) substantially determines the function of the polypeptide or polynucleotide. A sequence that has been modified to retain. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or mutation of at least one residue present in a naturally occurring protein.

  The term “derivative” as used herein with respect to a protein or polypeptide of the invention is used as long as the resulting protein or polypeptide substantially retains at least one of its intrinsic functions. Including substitution, mutation, modification, replacement, deletion, and / or addition of one (or more) amino acid residues from or to that sequence.

  The term “analog” as used herein with respect to a polypeptide or polynucleotide refers to any mimetic, ie, at least one of the intrinsic functions of the polypeptide or polynucleotide that it mimics. Includes compounds that possess one.

  Typically, amino acid substitutions can be made, for example from 1, 2, or 3 to 10 or 20 substitutions, provided that the modified sequence retains substantially the required activity or ability. Amino acid substitutions can include the use of non-natural analogs.

  Proteins used in the present invention can have amino acid residue deletions, insertions, or substitutions that result in silent mutations resulting in functionally equivalent proteins. As long as the intrinsic function is retained, deliberate amino acid substitutions can be made based on similarity of residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups that have similar hydrophilicity values Include asparagine, glutamine, serine, threonine, and tyrosine.

本明細書で使用される「ホモログ」という用語は、野生型アミノ酸配列および野生型ヌクレオチド配列と一定の相同性を有するものを意味する。「相同性（homology）」という用語は「同一性（identity）」と同じと考えられる。 For example, conservative substitutions can be made according to the table below. Amino acids in the same block in the second column, preferably amino acids in the same row in the third column can be substituted for each other:

As used herein, the term “homologue” means one having a certain homology with wild-type amino acid sequences and wild-type nucleotide sequences. The term “homology” is considered the same as “identity”.

  Homologous sequences are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% identical to the sequence of interest, preferably at least 95% or 97% or 99 Amino acid sequences considered to be% identical can be included. Typically, a homolog is considered to contain the same active site as the amino acid sequence of interest. While homology can also be considered in terms of similarity (ie, amino acid residues having similar chemical properties / functions), for the purposes of the present invention, homology is preferably represented by sequence identity.

  Homologous sequences are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% identical to the sequence of interest, preferably at least 95% or 97 It may contain nucleotide sequences that can be said to be% or 99% identical. Although homology can be considered from the viewpoint of similarity, in the context of the present invention, homology is preferably represented by sequence identity.

  Preferably, reference to a sequence having percent identity to any one of the SEQ ID NOs detailed herein refers to the sequence having the percent identity described over the entire length of the referenced SEQ ID NO. Say.

  Homology comparisons can be made visually or, more generally, using readily available sequence comparison programs. This commercially available computer program can calculate percent homology or identity between two or more sequences.

  Percent homology can be calculated over a contiguous sequence, i.e., aligning one sequence with the other sequence so that each amino acid in one sequence directly corresponds to the corresponding amino acid in the other sequence. And compare one residue at a time. This is called “no gap” alignment. Typically, such ungapped alignments are performed only with a relatively short number of residues.

  This is a very simple and consistent method, but, for example, when performing global alignments, a subsequent codon in a sequence pair in which one insertion or deletion in the nucleotide sequence is otherwise identical is used. It cannot be taken into account that it can be out of alignment and result in a significant reduction in percent identity. For this reason, most sequence comparison methods are designed to provide optimal alignments that take into account the possibility of insertions and deletions without penalizing the overall homology score excessively. This is accomplished by inserting “gaps” in the sequence alignment in an attempt to maximize local homology.

  However, these more complex methods assign a “gap penalty” to each gap that occurs in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible is highly related between the two compared sequences. Reflecting gender, you will achieve a higher score than one with many gaps. “Affine gap costs” are commonly used, which cost a relatively high cost to the existence of a gap and impose a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course result in optimized alignment with fewer gaps. Most alignment programs can change the gap penalty. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for gaps and -4 for each extension.

  Therefore, to calculate the maximum percentage homology, it is first necessary to create an optimal alignment taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A .; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software that can perform sequence comparison include BLAST package (see Ausubel et al. (1999) supra-Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol 403-410), as well as a set of comparison tools GENEWORKS, but not limited to them. Both BLAST and FASTA can be used for offline and online searching (Ausubel et al. (1999) supra, pages 7-58 to 7-60). However, depending on the application, it is preferable to use the GCG Bestfit program. Another tool called BLAST 2 Sequences can also be used for protein and nucleotide sequence comparison (FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187- (See 8).

  Although the final percentage homology can be assessed in terms of identity, the alignment process itself is usually not based on an all-or-nothing pair comparison. Nevertheless, a scaled homology score matrix is commonly used, which assigns a score to each pair-wise comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is often used is the BLOSUM62 matrix-the default matrix for the BLAST program suite. GCG Wisconsin programs generally use either public default values or, if specified, custom symbol comparison tables (see the user manual for more details). Depending on the application, it is preferable to use the public default values of the GCG package, or a default matrix such as BLOSUM62 for other software.

  Once the software has produced an optimal alignment, percentage homology, preferably percentage sequence identity, can be calculated. The software typically does this as part of the sequence comparison and produces a computational result.

  Although a “fragment” of full-length CNTF is also a variant, the term typically refers to a selected region of a polypeptide or polynucleotide of interest as a function or, for example, in an assay. Thus, “fragment” means an amino acid or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.

Such variants can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. When inserting, a synthetic DNA encoding the insert with the 5 'and 3' flanking regions corresponding to the naturally occurring sequence at both ends of the insertion site can be made. The flanking region can contain easy-to-use restriction enzyme sites that correspond to sites in the naturally occurring sequence, so that the sequence can be cleaved with the appropriate enzyme (s) It can be ligated to the cutting part. DNA is then expressed according to the present invention to produce the encoded protein. These methods are merely illustrative examples of the many standard techniques known in the art for manipulating DNA sequences, and other known techniques can be used.
Codon Optimization The polynucleotides used in the present invention can be codon optimized. Codon optimization has already been described in WO 1999/41397 and WO 2001/79518. Different cells differ in the frequency of use of specific codons. These codon biases correspond to biases in the relative abundance of specific tRNAs in the cell type. Expression can be increased by changing codons within the sequence and adjusting for the relative abundance of the corresponding tRNA. Similarly, expression can be reduced in a particular cell type by deliberately selecting codons that are rarely known for the corresponding tRNA. In this way, an additional level of translational regulation can be utilized.
(Example)

Materials and methods
Mice and anesthesia All animal experiments are conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, with valid UK Home Office approval. Went and got. A rhodopsin knockout mouse (C57B / 6.129 Rho ^tm1Phm , referred to herein as Rho ^-/- ) has been described (Humphries, MM et al. (1997) Nat. Genet. 15: 216-219), Jane Farrar , Trinity College Dublin, Ireland. An OPN1LW-EGFP mouse (C57BL / 6J ^{TgOPN1LW-EGFP85933Hue} , referred to herein as B6 ^{TgOPN1LW-EGFP} ) has already been described (Fei, Y. et al. (2001) Vis. Neurosci. 18: 615-623 This was obtained from Mutant Mouse Regional Resource Centers (MMRRC), National Institute of Health, USA, with the efforts of Dr. Rachel Pearson, University College London (UCL) Institute of Ophthalmology, London, UK. Mice that express EGFP in cone photoreceptors and have rod-specific primary degeneration (Rho ^{-/-TgOPN1LW-EGFP +/-} ) are ^homozygous for B6 ^{TgOPN1LW-EGFP + / +} mice (OPN1LW-EGFP transgene insertion) ) And Rho ^{− / −} (homotype for target rhodopsin knockout) and then backcrossed F1 progeny (Rho ^{+/− TgOPN1LW-EGFP +/−} ) with the parental Rho ^{− / −} strain. Mice were phenotyped by AF imaging at weaning and identified by PCR. All mice were housed on a standard 12 hour / 12 hour light / dark cycle with free access to food and water. General anesthesia is introduced by a single intraperitoneal injection of domitol (medetomidine hydrochloride, 1 mg / kg body weight) and ketamine (60 mg / kg body weight), with 1% tropicamide and 2.5% phenylephrine hydrochloride eye drops (both Bausch & Lomb , Kingston-Upon-Thames, UK). Anesthesia is abolished after treatment with an intraperitoneal injection of antisedan (Atipamezole, 5 mg / kg body weight) as necessary.
Vector Construction Adeno-associated virus serotype 2 vector backbone plasmid (pUF11) was provided by William W. Hauswirth, University of Florida, USA. Human nerve growth factor (NGF; NM_002506) and human ciliary neurotrophic factor (CNTF; NM_000614) cDNA clones were purchased from Origene (Rockville, MD, USA). NGF secretion signal using PCR (sense primer, GTAC GCGGCCGC ATGTCCATGTTGTTC; antisense primer, GCTCTGTGAAAGCTGAGTGTGGTTCC), and peripheral sequence with identity to CNTF coding region minus translation start codon and kozak consensus sequence (sense primer, GGAACCACACTCAGCTTTCACAGAGC; antisense primer , TCTA GTCGAC CTACATTTTCTTG) was amplified from each construct. The two fragments were then ligated using the “Swift PCR to ligate exons constructed in vitro” (SPLICE) reaction (Davies, WL et al. (2007) Biotechniques 43: 785-789) The AccI restriction enzyme site (underlined) was used to ligate to the pUF11 backbone. The fidelity of the final AAV2.CBA.hCNTF construct was confirmed by full-length sequencing in both directions.
Recombinant AAV serotype 2 (rAAV2 / 2) packaged CNTF construct by transient cotransfection of HEK293 cells seeded in virus production and titration cell factory (HYPERflask; Corning, Tewksbury, MA, USA) Virus was generated (referred to as rAAV2 / 2.hCNTF) and then as previously reported (Zolotukhin, S. (2005) Hum. Gene Ther. 16: 551-557; Jacobson, SG et al. (2006) Mol. Ther 13: 1074-1084), purified by iodixanol gradient centrifugation. Purified rAAV2 / 2.hCNTF virus was concentrated by buffer exchange (Amicon Ultra-15, Millipore, Billerica, MA, USA), which removes residual iodixanol and then sterile phosphate buffered saline The suspension was resuspended in water (PBS) to a total volume of 100 μL, and dispensed into preblocked tubes (0.01% BSA). Viruses are titrated by quantitative PCR (qPCR) using primers designed to amplify a 120 bp fragment within the polyA region using a vector plasmid and virus with known titers as a reference. did. The rAAV2 / 2.hCNTF virus titer was calculated as 2 × 10 ¹³ genomic particles (gp) / mL.
Intraocular injection
24-week-old Rho ^{-/-TgOPN1LW-EGFP +/-} mice with dilated pupil as described above, applied with gel lubricant (Viscotears, Novartis, Frimley, UK), then covered with cornea and 6 mm round A cover glass was placed to allow full visualization of the retina when viewed under a surgical microscope (M620 F20, Leica, Wetzlar, Germany). Injection was performed by advancing a Hamilton syringe with a 10 mm 34-gauge needle (65N, Hamilton AG) through the sclera and through the retinal neurosensory epithelium into the vitreous. 2 μL of vector suspension or buffer (PBS) was administered into the vitreous cavity near the posterior pole; reflux was minimized by normalizing intraocular pressure before removing the needle. Eye orientation and stability were maintained throughout by holding the superior rectus muscle with knurled forceps.
Enzyme-linked immunosorbent assay (ELISA)
HEK293 cells were seeded in 6-well plates (1 × 10 ⁶ cells / well) and transduced with 2 μL rAAV2 / 2.hCNTF (1 × 10 ¹³ gp / mL) virus. Three days after transduction, the medium was collected and assayed using a commercially available human CNTF specific ELISA (R & D systems, Abingdon, UK) according to the manufacturer's instructions. in vivo evaluation of hCNTF expression, 2x10 ⁸ gp (low dose), 2x10 ⁹ gp (medium dose) or with 2x10 ¹⁰ gp (high dose) rAAV2 / 2.hCNTF vector, old 4 weeks (PW4) Rho ^{- / -TgOPN1LW-EGFP +/-} mice were injected intravitreally. Since 4 weeks were allowed for genomic integration and vector expression, eyes were removed at that time (PW8) and snap frozen in liquid nitrogen. Eyeballs were thawed and homogenized in 100 μL PBS and hCNTF protein levels were assessed as described above using a commercially available human CNTF specific ELISA (R & D systems, Abingdon, UK).
8, 10, 12, 15, 18, 21 as described above using autofluorescence (AF) imaging and cone quantitative confocal scanning laser ophthalmoscope (cSLO; Spectralis HRA, Heidelberg Engineering, Heidelberg, Germany) At 24, 30 weeks, the fundus of each mouse was imaged (Charbel Issa, P. et al. (2011) Optimization of in vivo confocal autofluorescence imaging of the ocular fundus in mice and its application to models of human retinal degeneration. Invest. Ophthalmol. Vis. Sci.). Briefly, after dilation of the pupil, a contact lens is placed on the cornea using a viscous coupling gel (0.3% w / v hypromellose, Matindale Pharmaceuticals, Romford, UK) to prevent the formation of turbidity and image quality Improved and standardized. The mouse was placed on an imaging platform and the camera could be aimed at the focal plane of the retinal neurosensory epithelium using near infrared (NIR) spectroscopy mode (820 nm laser). Cone photoreceptors that originally express EGFP are imaged by autofluorescence (AF) mode (480 nm excitation) using a 55 ° lens, and high-resolution images (1536 x 1536 pixels) are automatically real-time (ART) Recorded with standard detection sensitivity by the mean method. The number of cones was manually quantified from each image using ImageJ software (Abramoff, MD et al. (2004) Biophotonics International 11: 36-42), but for each eye, over time. In order to normalize the area, vascular markings of the lower retina were used.
Electroretinogram (ERG)
ERG recording was performed as described above using the Espion E2 system (Diagnosys LLC, Cambridge, UK) according to standard protocols at PW8, PW10, and PW12 (Lipinski, DM, et al. (2011) Invest. Ophthalmol. Vis. Sci. 52: 6617-6623). After a period of light adaptation (> 10 minutes, multicolor white light, 30 cd / m ² ), a short (4 ms) white flash at 3, 10, and 25 cd.s / m ² (20 flashes per intensity) The b-wave amplitude was quantified using Espion software (Diagnosys LLC, Cambridge, UK).
Optomotor response (OMR)
A custom-made visual motion system was constructed, consisting of a rotating cylinder that allows precise control of speed and grid. The mouse was placed on a higher platform in the center of the drum, illuminated from above with a bright white stimulus (~ 1000 lux), and allowed to acclimate to the drum for 1 minute, while the drum remained stationary, and the mouse was free to explore the platform. We were able to. Each experiment consisted of 1 minute clockwise and 1 minute counterclockwise divided into alternating 30 seconds. The experiment was repeated three times independently at each time point for each animal, and the number of reactions was averaged. Although the rotation of the square wave grating causes a head tracking reaction, one reaction consists of a slow head tracking operation in the direction of drum rotation, followed by a reaction in which the head rapidly returns to the center position. The behavior of each mouse responding to the rotating drum was recorded using a digital camera mounted directly above the central platform; the number of head tracking reactions was manually performed using a blinded recorder for processing. I counted.
Laser speckle cortical imaging The cortex of 30-week-old Rho ^{-/-TgOPN1LW-EGFP +/-} mice that had received rAAV2 / 2.hCNTF treatment in one eye and PBS sham in the other eye (n = 4, high dose; n = 6, medium dose; n = 8, low dose). The positive subject group consisted of mice (n = 3) with the same background at a time when cone degeneration has not yet begun (6 weeks of age). Mice were dark acclimated for 8 hours prior to imaging, mice anesthetized with all preparatory steps under dark red illumination, and the pupils were fully dilated as described above. The head was fixed to a stereotaxic frame, the scalp was excised, and the skull covering the visual cortex was exposed. Transcranial imaging was performed using a Speckle Contrast Imager (moorFLIP, Moor instruments, Delaware, USA) and data was acquired at 25 Hz. Deep body temperature was monitored by rectal temperature measurement and maintained throughout with a feedback thermal pad that encased the experimental animal body. After each eye was stimulated independently, changes in cortical blood flow (CBF) were measured, but the opposite eye was always covered to maintain dark adaptation. The first eye to be examined was randomly chosen for each mouse, but the investigator blinded which eye received rAAV.hCNTF treatment. Each eye was stimulated by a short (10 ms) flash of 14.5 cd 510 ± 3 nm monochromatic light, 5 Hz for 1 s (Grass PS33 Photic Stimulator with LED stimulation). Stimulation was repeated 10 times with a 30 second interval between each eye (5 minutes total). The light stimulus immediately moved to the contralateral eye for examination, while the mouse and imaging device remained stationary. Data was processed using MATLAB R2012b (version 8.0.0.783). After downsampling the data to 5 Hz, a region of interest (ROI) was selected for the bilateral visual cortex, from which time series changes were extracted and averaged for the pixels within each ROI. Individual time series were flattened using a first kind Chebyshev filter. The percentage change in CBF over the 10 s prestimulation baseline period was calculated for the left and right hemispheres. The responses of both hemispheres were summed for each eye experiment (contralateral + ipsilateral) and the change in CBF size was compared between rAAV.hCNTF treated and PBS sham treated eyes.
RNA sequencing and analysis After euthanasia, mouse eyes were removed, placed in RNAlater (Ambion, Paisley, UK), snap-frozen on liquid nitrogen and stored at -80 ° C until processing. Total RNA was extracted by homogenizing the entire eye (RNA Tissue Mini Kit, QIAGEN, Manchester, UK). Contaminating DNA was removed by deoxyribonuclease I treatment according to the “on-column” digestion protocol (RNase-Free DNase, QIAGEN, Manchester, UK). Polyadenylated mRNA was purified from 2 μg total RNA per sample by magnetic separation (NEBNextPoly (A) mRNA Magnetic Isolation Module, New England Biolabs, Hitchin, UK). The library preparation protocol was performed using the NEBNext mRNA Library Prep Master Mix Set for Illumina Kit (New England Biolabs, Hitchin, UK) with the following changes: Additional purification (Ampure XP, volume ratio 2.8x, Ambion, Paisley, UK) and washing (2 x 80% ethanol) steps. Reverse transcription (RT) was performed using Superscript II (Invitrogen, Paisley, UK), followed by post-RT purification (Ampure XP, volume ratio 1.2 ×) and washing (2 × 80% ethanol) steps. The cDNA was end-modified, A-tailed, adapter ligated (custom adapter), and PCR amplified (12 cycles). The prepared library was multiplexed (4x) and evaluated for quality, followed by paired end sequencing of the IlluminaHiSeq 2000 platform with a 50 bp read length. Assess the quality of the output FASTQ file (SAMStat and FastQC: Read QC) and format it before adapter removal (Cutadapt) (FASTQ groomer; Blankenberg, D. et al. (2010) Bioinformatics 26: 1783-1785 ) And the paired-end method (TopHat for Illumina; Trapnell, C. et al. (2009) Bioinformatics 25: 1105-1111), and was aligned with the reference genome (Mus musculus, UCSC, mm9.fa). The output of recognized hits was converted to SAM format (SAM Tools) and hits were assigned to genomic features by comparison with a reference genome using HiSeq-Count. Using the DESeq R package (Bioconductor), the differential expression was invoked by normalization to the effective library size and the false discovery rate (FDR) was set to 2%. Genes with differences in expression were considered to have minimal changes in expression levels by a factor of 2 (12 log times). The heatmap output was created using RColorBrewer and R gplots packages.
The Anderson-Darling test for normality of the largest data set for each of the statistical data revealed that the data was extracted from a normally distributed population when: Cone quantification, A ² = 0.509, p = 0.1983; Low dose ERG, A ² = 0.3402, p = 0.4824; Medium dose ERG, A ² = 0.4483, p = 0.2785; High dose ERG, A ² = 0.7228, p = 0.0594. A plot of residuals versus the mean value fitted to each experimental group showed that the variance was equal within each group (plot not shown). In vivo cone quantification was analyzed using repeated measures two-way ANOVA with dose (independent variable) and time (repeated measurement) as factors and cone number as a dependent variable. Using repeated measures two-way ANOVA, we analyzed the ERG response using flash intensity (independent variable) and time (repeated measurement) as factors, and in each case using the bright spot b-wave amplitude as a dependent variable. The dose was analyzed independently. Normality was not confirmed for optomotor response data: A ² = 4.5536, p = <0.0005. A non-parametric Kruskal-Wallis test was performed with Dunn's multiple comparison test to compare the mean head tracking response elicited from treated and untreated eyes at each dose. Due to the small sample size, variance could not be plotted for groups when assessing CNTF levels after in vitro or in vivo vector transduction. A non-parametric Mann-Whitney U test was applied to two examples to compare mean CNTF levels in transduced cell supernatants for mock transduction (in vitro) or in the isolated eye (in vivo). Bonferroni post test was applied to all analysis of variance. The significance (p) level for all tests was set to 0.05. Control. ns = not significant, *** = p <0.001, one-way analysis of variance. Low dose n = 5, medium dose n = 6, high dose n = 4.
Results To accurately model cone survival and function in response to long-term CNTF treatment, C57B / 6.129 Rho ^tm1Phm mice homozygous for rhodopsin (Rho) knockout are highly sensitive green in cone photoreceptors. ^Crossed with C57BL / 6J ^{TgOPN1LW-EGFP85933Hue} transgenic mice expressing fluorescent protein (EGFP) (Fei, Y. et al. (2001) Vis. Neurosci. 18: 615-623; Humphries, MM et al. (1997) Nat. Genet. 15: 216-219). In this end stage retinitis pigmentosa (RP; name here, Rho ^{-/-TgOPN1LW-EGFP +/-} ) model, apoptosis of rod photoreceptors due to the absence of rhodopsin protein is postnatal (PW) It appears by 8 weeks, followed by a secondary loss of autofluorescent cones, leading to global photoreceptor degeneration (Hobson, AH et al. (2000) Exp. Eye Res. 71: 247 -254). The use of an animal model in which cone photoreceptors degenerate following an advanced rod reduction reflects the disease phenotype most commonly seen in RP patients (rod-cone dystrophy). In addition, this model closely replicates the clinical scenario where neuroprotection is most likely applied, with most of the rod photoreceptors degenerated, but the priority is to lose the photoreceptor cells. Regardless of the etiology, it is to protect the eyesight of the cone. This approach has particular implications for RP, and the cause of photoreceptor loss is 50% in autosomal dominant cases and 30% in autosomal recessive cases, which are not fully understood (Hartong, DT et al. (2006 ) Lancet 368: 1795-1809; Lipinski, DM et al. (2013) Prog. Retin. Eye Res. 32: 22-47).

A vector based on recombinant adeno-associated virus serotype 2 (rAAV2 / 2) was made (1x10 ¹³ genomic particles (gp) / mL) and modified for extracellular release by addition of upstream human nerve growth factor secretion signal Human CNTF (hCNTF) protein was expressed. Protein secretion from the resulting rAAV2 / 2.hCNTF vector was verified in vitro by HEK293 cell transduction (MOI = 1000), but in the medium by enzyme-linked immunosorbent assay (ELISA; Figure 6a). Results in high levels of hCNTF protein being detected. In vivo administration of rAAV2 / 2.hCNTF (intravitreous injection) into the vitreous cavity of PW4 Rho ^{-/-TgOPN1LW-EGFP +/-} mice (n = 3 per group) similarly produced high levels of secreted hCNTF protein. However, it is most likely from ganglion and Mueller glial cells, which are known to be efficiently transduced by AAV2-based vectors. Importantly, it was confirmed that the effective amount of hCNTF protein can be controlled extensively by changing the number of genomic particles administered per eye (FIG. 6b).
CNTF prevents the development of RP in a dose-dependent manner
Rho ^{− / − TgOPN1LW-EGFP +/−} mice were treated with low dose (2 × 10 ⁸ gp; n = 8 mice), medium dose (2 × 10 ⁹ gp; n = 6) before cone photoreceptors were significantly lost (PW4). Mice) or high doses (2 × ^{10 10} gp; n = 5 mice) received intravitreal injections of rAAV2 / 2.hCNTF. In all mice, the contralateral eye received a sham injection of the same volume (2 μL) of phosphate buffered saline (PBS) to serve as a control for the effects of surgical intervention.

Four weeks after injection (PW8), the retinas of the injected mice were imaged with a confocal scanning laser ophthalmoscope (cSLO), which was non-invasive of retinal lesions by directly visualizing the fundus through the pupil. Enable evaluation. Specifically, cSLO autofluorescence (AF) imaging mode utilizes a laser with an excitation wavelength closest to the absorption peak of EGFP, which allows visualization of EGFP-expressing cells in the retina (Beck, SC et al. al. (2010) Invest. Ophthalmol. Vis. Sci. 51: 493-497; Charbel Issa, P. et al. (2011) Optimization of in vivo confocal autofluorescence imaging of the ocular fundus in mice and its application to models of human retinal degeneration. Invest. Ophthalmol. Vis. Sci.). AF imaging of the Rho ^{-/-TgOPN1LW-EGFP +/-} mouse fundus showed a dot-like pattern (Figure 1a) corresponding to the expected distribution of EGFP-expressing pyramidal photoreceptors throughout the retina. ^Post- mortem lectin staining of retinas detached from Rho ^{-/-TgOPN1LW-EGFP +/-} mice immediately after AF imaging (Fig. 1c) shows that individual "dotted" signals observed in vivo, each expressing EGFP It was confirmed that it corresponds to a cone photoreceptor (FIGS. 1a-c). Accurately quantitate cone photoreceptors using vascular labeling to designate a standard region of interest in each eye that will not change throughout the life of the experimental animal and survive in response to hCNTF treatment Could be evaluated in the long term (Fig. 1d-g; Fig. 6c-j).

AF imaging showed an equivalent distribution of EGFP expressing cone photoreceptors at PW8 in all treatment and control groups (FIG. 1h; FIGS. 7a-d). The number of cone photoreceptors dropped rapidly during the time course of the experiment in the PBS sham group, and the number of cones reached zero in PW24 (FIG. 1h, FIG. 7d). Consistent with the absence of detectable hCNTF protein (Figure 6b), the number of pyramidal photoreceptors in the eye that received intravitreal injections of low dose (2x10 ⁸ gp) rAAV2 / 2.hCNTF was paired with PBS. There was no significant difference at any time point for eyes receiving sham injections (FIG. 1h; FIG. 7c; p ≧ 0.05, repeated measures two-way analysis of variance with Bonferroni post-test, n = 8). Near-infrared spectroscopy (NIR) imaging showed a geographic change in lesions in the retinal pigment epithelium (RPE), consistent with the disappearance of overlying photoreceptors, a common clinical finding of terminal RP (Figure) 2a-d, Fig. 7c-d). This finding was supported by histological studies performed at PW30, which indicated the absence of both rod and cone photoreceptors in PBS sham-treated eyes (FIG. 2i).

The number of cones in eyes treated with medium dose (2x10 ⁹ gp) or high dose (2x10 ¹⁰ gp) rAAV2 / 2.hCNTF vector was significantly greater than PW12 compared to contralateral control eyes ( FIG. 1h; FIG. 7a-b). Neuroprotection of cone photoreceptors is dose-dependent, with maximum survival observed in high dose treated eyes (62.38 ± 1.95%) compared to medium dose treated eyes (44.90 ± 1.69%; PW30 vs. PW8) It was done. The rate of cone photoreceptor loss was close to zero after PW15 in both treatment groups (FIG. 1h; 0.42-0.69% / week); no RP phenotype was seen in medium or high dose treated eyes (Fig. 2e-h; Fig. 7a-b). Histological examination in PW30 revealed that both cone (GFP-expressing) and rod (GFP non-expressing) photoreceptors are preserved in eyes receiving high-dose rAAV2 / 2.hCNTF vector (Figure 2j). The lack of co-localization of EGFP with medium-wavelength sensitive (Mws) cone opsin expression strongly suggests the presence of isolated cone outer segments (Fig. 2k-o), and the presence of its associated cilia In some cases, it effectively prevents transport of EGFP protein from the photoreceptor outer segment.
CNTF protects visual function despite mild suppression of electrophysiological responses
rAAV2 / 2.hCNTF-treated mice underwent electroretinography (ERG) at PW8, using corneal electrodes to indirectly measure the electrical response of the retina after light stimulation. Technology. Consistent with previous studies demonstrating reduced cone sensitivity in the presence of CNTF protein, it was observed that cone photoreceptor response was dose-dependently suppressed (Wen, R. et al (2012) Prog. Retin. Eye Res. 31: 136-151) (FIGS. 8a-f). ERG peak recording was considered unrecordable because it was below the average amplitude of background noise (˜20 μV) in all groups until PW12. The decrease in ERG peak amplitude occurred at the same time as the significant loss of cone photoreceptors in all groups, but eyes that received medium dose (2x10 ⁹ gp) and high dose (2x10 ¹⁰ gp) treatment It may show significant protection of the cone past PW12, indicating that there is no cone vision despite protection of the cone photoreceptor (Fig. 2j-o). Therefore, further electrophysiological examinations and behavioral experiments were performed at PW30 to verify the visual function in more detail.

The ability of the remaining cones to transmit signals through the visual pathway to the brain was evaluated by performing laser speckle imaging (LSI) of the visual cortex during retinal stimulation with light. LSI is a non-contact imaging technique with high spatio-temporal resolution that can detect random speckle pattern changes linearly related to the increase in red blood cell movement in microvessels (Figure 3c). In this specification, LSI was used to measure changes in cortical blood flow (CBF) in the visual cortex after monocular stimulation with a short-time (10 ms) flash of 14.5 cd monochromatic light (510 ± 3 nm). Its wavelength is closest to the maximum absorption peak of the mouse M-cone. The ability of selected stimulation parameters to cause a cone-mediated response was verified for ^{PW6 naïve} Rho ^{− / − TgOPN1LW-EGFP +/−} mice (n = 3). CBF changes were mainly detected in the visual cortex on the opposite side of the stimulated eye, indicating proper downstream processing of the visual stimulus (Figure 3a-c). When stimulating eyes treated with medium dose (2x10 ⁹ gp) rAAV2 / 2.hCNTF vector, PW30 cortical LSI revealed increased CBF changes in the contralateral visual cortex, but not significantly ( Figure 3d-f; p = 0.20, one-sided Wilcoxon signed rank test, n = 6). In contrast, LSI when stimulated eyes receiving high dose (2x10 ¹⁰ gp) rAAV2 / 2.hCNTF demonstrated an increase in contralateral visual cortex, which stimulated sham-treated control eyes It was significantly greater than the response sometimes elicited (FIG. 3c, gi; p = 0.012, one-sided Wilcoxon signed rank test, n = 4). When compared to baseline LSI CBF measurements obtained from PW6 animals (+ 0.819%), the magnitude of CBF changes (+ 0.509%) induced after stimulation of high-dose CNTF-treated eyes was observed in PW30 Were largely correlated with the percentage of cone photoreceptor survival (62.38 ± 1.95% versus PW8). Cortical LSI strongly showed that the living cones were still light sensitive and could transmit signals to the brain. The degree to which changes in cortical blood flow correspond to practical visual acuity processing was assessed by optomotor response (OMR) measurements. OMR measures the number of unintentional head tracking movements that an experimental animal performs in response to a visual stimulus in the entire field of view, which is classically defined as a constant width and contrast in the vertical direction (100% contrast; 0.2 cycles / Consists of a brightly illuminated (~ 1000 lux) rotating drum with black and white stripes, with animals in it (Figure 4a). OMR is characterized by a slow phase head tracking operation in the direction of drum rotation, followed by a rapid phase change of the head to a central position. Slow phase head tracking motion is triggered independently by each eye, but it depends on the direction of drum rotation (Fig. 4a) (Hobbelen, JF et al. (1971) Doc. Ophthalmol. 30: 227-236; Harvey, RJ et al. (1997) Vision Res. 37: 1615-1625; Douglas, RM et al. (2005) Vis. Neurosci. 22: 677-684). When studying experimental animals at PW30, the number of head tracking reactions per minute recorded from sham-operated eyes did not significantly exceed zero in any group (0.33 ± 0.05 / min). The number of head tracking responses caused by stimulation of medium dose (2.11 ± 0.31 / min) and high dose (6.92 ± 0.92 / min) treated eyes is significantly greater than paired sham-treated eyes (FIGS. 4b-c; p = <0.001, Kruskal-Wallis test, Dunn's multiple comparison method, n = 4), linear regression analysis of the OMR response to the number of remaining cones in PW30 revealed a significant positive correlation (r ² = 0.80 , F = 33, p = 0.0004) (FIG. 9a).

Together, these data show that cone photoreceptors preserved by hCNTF neuroprotection are still sensitive to light, despite the apparent suppression of electrophysiological function as assessed by ERG. It strongly suggests that it has the ability to effectively transmit signals to the optic nerve pathway via the optic nerve.
CNTF upregulates complement factor proteins and proteolytic inhibitors
We worked on transcriptome analysis of 23,365 mouse genes to determine the mechanisms underlying long-term neuroprotection of cone photoreceptors in response to CNTF. Next generation sequencing of total RNA extracted from whole eyes of PW30 compared the treated eye transcriptome with paired sham-treated controls (n = 4 eyes for each group) at each dose. . According to the Benjamini-Hochberg method, the false discovery rate was fixed at 2% and all samples were normalized to the effective library size. When comparing the transcriptome of high-dose treated (2 × ^{10 10} gp) eyes with a paired PBS sham-treated control, it was found that 1,533 genes showed significant differential expression variation (p <0.05; FIG. 9b). ). Of these, 460 genes showed> 2-fold change in expression level, and were then classified by cell function using gene ontology (FIGS. 5a-b). Serine-, cysteine-, and metallo-type peptidase inhibitors (17 genes; Table 1.1) constitute the most up-regulated family of transcription and members are paired sham treatments in high-dose hCNTF-treated eyes The expression was up to 89 times higher than the control. Members of this group encode intracellular and extracellular peptidase inhibitors, whose dysfunction has previously been linked to the development of retinal diseases; such inhibitors include, for example, tissue inhibitors of metalloproteinase 3 ( Timp3, +3.49 fold), and the mutation is associated with autosomal dominant sourceby fundus degeneration (Weber, BH et al. (1994) Nat. Genet. 8: 352-356). Transcriptional regulation of peptidase inhibitors is not well understood, but activation is via signal transduction transcription factor 3 (Stat3) (+2.46 fold expression) and a member of the CCAAT / enhancer binding protein (Cebp) family of transcription factors 3 of them (Cebpα, Cebpβ, and Cebpδ) were also significantly upregulated (Table 1.2) (Zhao, H. et al. (2007) Mol. Cell Biol. 27: 5286-5295; Udofa, EA et al. (2013) PLoS One 8: e57855).

  Several members of the classical and secondary complement cascades (16 genes, Table 1.3) were significantly up-regulated (up to 10-fold), including various complement components (eg C3 , C4a) and complement factor genes (eg Cfb). Numerous modulators of the cytokine signaling system are up-regulated (19 genes, up to 8 fold; Table 1.4), which includes cytokine signaling system 3 suppressors (SOCS3) and also 5 suppressors (SOCS5) Although included, these are expressed in photoreceptors and are known to reduce stress-induced inflammatory responses (Ozawa, Y. et al. (2008) J. Biol. Chem. 283: 24561-24570; Takase, H. et al. (2005) J. Neuroimmunology 168: 118-127).

Downregulation (1.7x) of several genes encoding lecithin retinol acyltransferase (LRAT), an important component of the vitamin A cycle, and ion channels and G protein-coupled receptors expressed in internal retinal neurons Observed in medium, and high dose rAAV2 / 2.hCNTF-treated eyes (Table 1.5), which was consistent with previous studies and showed extensive changes in optical signaling in response to CNTF treatment (Wen, R. et al. (2012) Prog. Retin. Eye Res. 31: 136-151).
By taking advantage of the visual characteristics unique to the eye, progressive neurodegeneration can be assessed in real time and the survival of autofluorescent cone photoreceptors can be accurately quantified by repeated in vivo imaging. This study for the first time reveals the ability of neuroactive compounds to provide long-term protection in a degenerative disease model.

  The data in this study demonstrate that pyramidal photoreceptors are protected by hCNTF secretion by rAAV beyond the point where complete lateral retinal degeneration is expected without intervention. Stabilization of the number of cone photoreceptors was observed in the medium-dose (44.90 ± 1.69%) and high-dose (62.38 ± 1.95%) treatment groups, suggesting that protection may be maintained over time. These findings are important in light of recent clinical trials that use encapsulated cell technology to provide sustained release of CNTF, and this photoreceptor protection has been demonstrated in the short to medium term (up to 5 years) The possible consequences of long-term neuroprotection remain unknown due to the slow progression of RP in humans (Birch, DG et al. Long-term follow-up of patients with retinitis pigmentosa (RP) receiving sustained -release CNTF through intraocular encapsulated cell technology implants. ARVO annual meeting (Seattle, WA, 2013); Birch, DG et al. (2013) Am. J. Ophthalmol. 156: 283-292 e281).

  In this study, cell survival was observed in a dose-dependent manner, but the physiological inhibition of function assessed by electroretinography (ERG) was also observed; No dose was achieved to achieve neuroprotection (McGill, TJ et al. (2007) Invest. Ophthalmol. Vis. Sci. 48: 5756-5766). The number of photoreceptors protected and the sensitivity of each photoreceptor to light appears to be directly proportional to the bioavailability of the hCNTF protein. This strongly suggests that the mechanisms underlying functional suppression and cell survival are two sides of the same coin. Thus, when patients have good vision, the potential to extend vision beyond the point where they would lose vision if not treated can outweigh the suppression of visual function in the early stages of progressive degenerative disease There is sex.

  The time of intervention is another factor that is likely to be important for the successful protection and maintenance of cone photoreceptors. In this study, we aimed to construct a clinical scenario in which a significant proportion of rod photoreceptors disappeared at the time of maximum CNTF expression. This was achieved by deciding the timing of rAAV.hCNTF administration so that the peak of transgene expression does not appear by PW8-10, but by that time the rod degeneration was sufficiently degenerated in the rhodopsin knockout mouse model. It is ongoing (Humphries, MM et al. (1997) Nat. Genet. 15: 216-219). Although our findings are promising, the histological examination presented here clearly showed protection of rod photoreceptors (GFP non-expressing cells) in addition to cones after CNTF treatment .

  Interestingly, despite early intervention, past preclinical trials cannot uniformly observe functional protection and cannot maintain vision even if treated before the onset of retinal degeneration It was suggested that it might be. Consistent with such studies, we first used ERG as a primary assessment of remaining electrophysiological function and observed that measurements were not recordable in all groups by PW12. Since ERG is an indirect measurement of retinal function that relies on the propagation of electrical signals with sufficient amplitude and uniformity to be detected by electrodes placed on the corneal surface, the recordable cone ERG It was thought that losing too much does not necessarily reflect the lack of functional vision. In fact, a small area of the retina is light sensitive, but there is a good chance that it will not propagate a detectable signal, focusing on photoreceptor transplantation and optogenetic approaches to restore light sensitivity to the degenerated retina Research has best highlighted the principle, but in this case functional vision can be partially restored, yet the ERG response remains almost certainly absent (Mace, E et al. (2015) Mol. Ther. 23: 7-16; Gaub, BM et al. (2014) Proc. Natl. Acad. Sci. USA 111: E5574-5583; Singh, MS et al. (2013) Proc. Natl. Acad. Sci. USA 110: 1101-1106; Francis, PJ et al. (2013) Translational vision science & technology 2: 4).

  The factor causing the lack of ERG response observed past PW12 in this study may be suppression of outer segment formation, as described for rod photoreceptors after CNTF treatment (Wen, R. et al. (2006) J. Neurosci. 26: 13523-13530). Studies on the effect of CNTF on cone outer segment formation are limited; however, we observe a constant opsin-containing cone outer segment persistence in PW30, indicating that cones are ERG It strongly suggests that it may still be photosensitive despite the absence (Li, Y. et al. (2010) PLoS One 5: e9495; Beltran, WA et al. (2007) Exp. Eye Res. 84: 753-771; Rhee, KD et al. (2013) Proc. Natl. Acad. Sci. USA 110: E4520-4529). Laser speckle imaging (LSI) was performed to examine the hypothesis that cone photoreceptors retain the ability to relay interpretable signals to the visual and pretectal areas of the brain. LSI when stimulated on one side is a robust method for assessing the photosensitivity of the remaining cones, and, importantly, the responses from treated and untreated eyes in each animal are described separately. Enable. Cortical imaging clearly showed that stimulation of high-dose rAAV.hCNTF-treated eyes causes significant changes in cortical blood flow in the contralateral visual cortex, which shows that the remaining cone photoreceptors receive light It strongly suggests that it is possible and still correctly synaptically formed. Importantly, the propagation of optomotor responses indicated that the signal received by the visual cortex symbolizes physiologically relevant information. The head tracking response did not occur after stimulation of sham-treated control eyes, but this supports the evidence that endogenous photoreceptor retinal ganglion cells that are present in the inner retina even in end stage degeneration are not involved in OMR ( Cahill, H. et al (2008) PLoS One 3: e2055). The findings are also particularly relevant to clinical trials that express hCNTF using encapsulated cell technology, but despite short-term suppression, practical vision is still in end-stage disease and blindness occurs This is because it demonstrates that it may be preserved beyond a possible point in time.

  The impact of CNTF administration appears not only important in the treatment of RP, where photoreceptors are functionally suppressed with increasing doses, but is also an important consideration when evaluating treatments for other neurodegenerative diseases It is a matter. More specifically, previous unsuccessful clinical trials aimed at preventing the loss of motor neurons in ALS have no therapeutic effects and no adverse events in patients treated systemically with high doses of CNTF (A double-blind placebo-controlled clinical trial of subcutaneous recombinant human ciliary neurotrophic factor (rHCNTF) in amyotrophic lateral sclerosis. ALS CNTF Treatment Study Group. (1996) Neurology 46: 1244-1249; Bongioanni, P. et al. (2004) The Cochrane database of systematic reviews, CD004302). These findings indicate that a more targeted approach may be required than provided by current neuroprotection strategies, which enables effective mechanisms underlying cell survival to reduce side effects. Separated from the mechanism that brings it. In this study, the mechanism of neuroprotection via CNTF after sustained expression has not been fully elucidated so far, but transcriptome analysis of neurons surviving in end-stage retinal disease I was able to clarify my own insights. Transcriptome analysis of rAAV2 / 2.hCNTF-treated eyes on sham-treated eyes revealed several important results. First, significant overexpression of the Stat3 gene was observed in medium and high dose rAAV2 / 2.hCNTF-treated eyes, which is a receptor complex of Cntfr / Lifrβ / Gp130 activity in which CNTF is mediated by Stat3 (Rhee, KD et al. (2013) Proc. Natl. Acad. Sci. USA 110: E4520-4529; Davis, S. et al. (1993 Science 259: 1736-1739; Kassen, SC et al. (2009) Exp. Eye Res. 88: 1051-1064; Peterson, WM et al. (2000) J. Neurosci. 20: 4081-4090).

  Second, several gene families encoding inhibitors of proteolysis are strongly overexpressed (up to 89-fold), suggesting a major role in hCNTF-mediated neuroprotection. Activation of the serine protease inhibitor (Serpin) family (11 gene) members is particularly closely related to Stat3 expression levels and has been found to be anti-apoptotic when overexpressed in tumors ( Ahmed, ST et al. (2009) Biochem. Biophys. Res. Commun. 378: 821-825). Inhibitors of proteolysis may play a crucial role in retinal diseases, such as Timp3 and Serpin1b, which prevent the degradation of extracellular matrix components and Bruch's membrane, respectively. Acts and is involved in sourceby fundus degeneration and age-related macular degeneration (AMD) (Weber, BH et al. (1994) Nat. Genet. 8: 352-356; Chong, NH et al. (2005) Am J. Pathol. 166: 241-251; Sorsby, A. et al. (1949) Br. J. Ophthalmol. 33: 67-97). Since the physical degradation of neurons, such as photoreceptors, is typically caused by lysis through serine-cysteine proteases, lysosomal proteases, and complement in neurodegenerative diseases, the proteins observed herein It is postulated that overexpression of endogenous inhibitors of degradation may provide a definitive defense against neuronal apoptosis in response to cellular stress. It is suggested that direct overexpression of secreted proteolytic inhibitors may provide a new therapeutic tool to prevent neuronal cell death in degenerative diseases. In fact, using a targeted approach aimed at preventing photoreceptor degeneration in RP by directly inhibiting cell body and extracellular matrix degeneration can physiologically suppress function. It may be possible to protect photoreceptor cells without. In the case of ALS, directly preventing degeneration of cortical motoneurons by inhibiting proteolysis achieves cure without significant side effects observed in clinical trials when CNTF is administered systemically at high doses ALS CNTF Treatment Study Group. (1996) Neurology 46: 1244-1249; Bongioanni, P. A double-blind placebo-controlled clinical trial of subcutaneous recombinant human ciliary neurotrophic factor (rHCNTF) et al. (2004) The Cochrane database of systematic reviews, CD004302).

  Finally, a sustained innate immune response, characterized by significant up-regulation (up to 10-fold) of several members (16 genes) of the classical and secondary complement cascades, Observed in high-dose treated eyes, this includes two complement factors related to AMD (encoded by the Cfi gene) and retinal photodamage (encoded by the Cfd gene) (van de Ven, JP et al. (2013) Nat. Genet. 45: 813-817; Rohrer, B. et al. (2007) Invest. Ophthalmol. Vis. Sci. 48: 5282-5289). Interestingly, neither complement up-regulation nor cytokine up-regulation was observed in low dose treated eyes with PW30, indicating that the innate immune response ceases upon completion of photoreceptor degeneration. Although the role of some up-regulated protease inhibitors remains unclear, the expression of genes encoding proteolytic inhibitors is dependent on various components (eg, C3, C4, and Cfb proteins) after proteolytic cleavage It may also be particularly relevant to alteration of the innate immune response occurring in the central nervous system that is activated for the first time. For example, Serpin3k is known to have strong anti-inflammatory and anti-angiogenic effects, and by blocking the Wnt pathway, tumor necrosis factor α (Tnf-α), cell adhesion molecule 1 (Icam-1) and vascular endothelium Inhibits cell growth factor (Vegf) (Zhang, B. et al. (2009) Invest. Ophthalmol. Vis. Sci. 50: 3943-3952). In addition to preventing retinal necrotic cell death, Serpin3k also significantly inhibits rod outer segment formation, so its upregulation by hCNTF may contribute to reduced retinal sensitivity (Zhang, B. et al. (2009) Invest. Ophthalmol. Vis. Sci. 50: 3943-3952; Zhang, B. et al. (2010) Proc. Natl. Acad. Sci. USA 107: 6900-6905; Zhang, B. et al. (2008) PLoS One 3: e4077).

上記明細書に記載のすべての刊行物は、参照により本明細書に組み入れられる。記載されている本発明の化合物、使用、および方法のさまざまな修正および変更は、本発明の範囲および精神から逸脱することなく、当業者には明白であろう。本発明は、特定の好ましい実施形態に関連して説明してきたが、当然のことながら、請求される発明はそのような個別の実施形態に過度に制限されるべきではない。実際、記載されている本発明を実施するための方法のさまざまな修正が、生化学およびバイオテクノロジーまたは関連分野の当業者には明白であり、それらは下記の特許請求の範囲に含まれるものとする。 In summary, this study demonstrates that the optical properties unique to the eye allow neuronal survival in response to therapeutic intervention to be assessed in a long-term and very accurate manner through repeated in vivo imaging. Demonstrated. Herein, sustained hCNTF expression results in long-term preservation of cone photoreceptors, indicating that practical visual acuity is maintained until the end of degeneration despite slight electrophysiological suppression It was. This study showed that healthy cell survival is directly related to available doses of CNTF. Transcriptome analysis indicates that Stat3-mediated overexpression of intracellular and extracellular protease inhibitors underlies pyramidal protection in RP after hCNTF treatment, which probably directly inhibits cell degradation and extracellular matrix degradation It is strongly suggested that. We have shown that overexpression of certain endogenous protease inhibitors can provide a novel therapeutic tool for neuronal protection against cell death in neurodegenerative diseases, such as RP / AMD and other ALS. put forward.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described compounds, uses and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such individual embodiments. Indeed, various modifications of the described methods for carrying out the invention will be apparent to those skilled in biochemistry and biotechnology or related fields, which are intended to be within the scope of the following claims. To do.

Claims (15)

  A method of treating or preventing retinitis pigmentosa, wherein an adeno-associated virus (AAV) vector containing a nucleotide sequence encoding ciliary neurotrophic factor (CNTF) is administered to a subject in need thereof Said method.   2. The method of claim 1, wherein the AAV vector contains an AAV serotype 2 genome.   The method according to claim 1 or 2, wherein CNTF is human CNTF.   4. The method according to any one of claims 1 to 3, wherein the AAV vector contains a secretory signal sequence operably linked to a nucleotide sequence encoding CNTF.   5. The method of claim 4, wherein the secretion signal sequence is a human neurotrophic factor (NGF) secretion signal sequence.   6. The method of any one of claims 1-5, wherein the AAV vector is administered to the eye of the subject by subretinal injection, direct retinal injection, or intravitreal injection.   The method according to any one of claims 1 to 6, wherein the AAV vector is administered to the eye of the subject by subretinal injection.   8. The method of any one of claims 1-7, wherein the AAV vector is administered to the subject in a single dose.   9. The method of any one of claims 1-8, wherein the subject is substantially devoid of rod cells in the eye to be treated at the time of administration of the AAV vector.   10. The method according to any one of claims 1 to 9, wherein degeneration of photoreceptor cells due to retinitis pigmentosa is substantially prevented over the life of the subject.   11. The method of claim 10, wherein cone cell degeneration due to retinitis pigmentosa is substantially prevented over the life of the subject.   A method of reducing photoreceptor cell death in a subject suffering from or at risk of developing retinitis pigmentosa, comprising: ciliary neurotrophic factor (CNTF) Administering an adeno-associated virus (AAV) vector containing a coding nucleotide sequence to a subject in need thereof, wherein visual function is substantially restored or maintained in the treated eye, Said method.   13. The method of claim 12, wherein photoreceptor degeneration due to retinitis pigmentosa is substantially prevented over the life of the subject.   14. The method of claim 12 or 13, wherein degeneration of pyramidal cells due to retinitis pigmentosa is substantially prevented over the life of the subject.   15. A method according to any one of claims 12 to 14, wherein the subject is substantially devoid of rod cells in the eye to be treated at the time of administration of the AAV vector.

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