“Draft:Adeno associated virus and gene therapy of the human retina”的意思、由来-开放百科全书

Gene Therapy is the use of genetic material (DNA) inserted into a patient’s cell for the treatment of an inherited or acquired diseases. There are many medical conditions that are a result of mutation in patient’s gene. Gene therapy tries to introduce functional form of that gene into the patient’s cell nucleus in order to compensate for the mutated gene^[1]).

Retinal gene therapy holds great promise in treating different non-inherited and inherited blindness. The first gene therapy trial for inherited retinal disease took place in 2007 at Moorfield Eye Hospital and University College London’s Institute of Ophthalmology. This therapy was done to treat Leber’s congenital amaurosis diseases which is an inherited blinding disease caused by mutations in RPE65 gene. The result was somewhat promising, showing slight increase in vision and most importantly there were no side-effects^[2]

In retinal gene therapy the most widely used vectors for ocular gene delivery are based on adeno-assosciated virus. The great advantage in using adeno-associated virus for the gene therapy is that it poses minimal immune responses and mediates long-term transgene expression in a variety of retinal cell types. For example tight junctions that form the blood-retina barrier, separate subretinal space form the blood supply, providing protection from microbes and decreasing most immune-mediated damages.(2)

The human adeno-associated virus (AAV) is one of the smallest viruses with a non-enveloped icosahedral capsid of approximately 22 nm ^[3]^[4](3,4). It was discovered in 1965 as a contaminant of adenovirus (AD) preparations(3). The AAV has a linear single-stranded DNA genome that consists of 145 nucleotide-long inverted terminal repeat (ITR) and a size of approximately 4.7-kilobases (kb)(3,4). Since a virus does not encode a polymerase it has to rely on host cellular polymerase activities to replicate its DNA(3). The ITRs flank two viral genes called rep (replication) and cap(capsid)(3,4). The rep is composed of four overlapping genes encoding rep protein which is required for the AAV life cycleThe cap gene contains sequences of capsid proteins: virion protein 1 (VP1), VP2 and VP3, which ultimately forms a capsid of icosahedral symmetry(3,4).

An interesting property of the ITRs is that they can form a hairpin, which contributes to self-priming that allows primase-independent synthesis of the second DNA strand and they are also required for the integration of the AAV DNA into the host cell genome (3). The one side of the AAV genome contains two promoters at position 5 (p5) and 19 p(19)(3,4). These two promoters produce two overlapping messenger ribonucleic acids (mRNAs) and through the process of splicing different lengths of the (mRNAs) are produced(3). There are four different possibilities of mRNA products; four Rep proteins can be synthesized with different sizes: Rep78, Rep68, Rep52, and Rep40(3,4). The other side of the AAV genome contains one promoter at position 40 (p40) and produces sequences of three capsid proteins, VP1, VP2 and VP3(3,4).

The primary attachment site for AAV2 virions is the heparin sulphate proteoglycan and the process of internalization occurs with the help of co-receptors αvβ5 heterodimers, fibroblast growth factors receptor type 1 and the hepatocyte growth factor receptor, c-Met (4).

Once the AAV has entered the host cell, it can go through two different pathways of life cycle: the lytic or the lysogenic. The lytic stage occurs in cells infected with a helper virus such as Adeno virus (AD) or herpes simplex virus (HSV), whereas the lysogenic stage is established in host cells in the absence of (or, without) helper virus (4). During AAV infection of just a human cell, its gene expression program is auto-repressed and the virus genome integrates into a region of roughly 2-kb on the long arm of human chromosome 19 3,4). The level specificity during the process makes AAV biology very attractive for safe and stable transgene expression. The site-specific integration sites are composed in cis next to the ITRS. The ITRS sequences is required in cis to the therapeutic gene and structural (cap) and packaging (rep) genes can be delivered in trans(3,4).

One important factor in gene delivery is developing altered cell tropisms to narrow or broaden rAAV-mediated gene delivery and to increase its efficiency in tissues. Specific properties like capsid conformation, cell targeting strategies can determine which cell types are affected and also the efficiency of the gene transfer process. Different kinds of modification can be undertaken. For example modification by chemical, immunological or genetic changes that enables the AAV2 capsid to interact with specific cell surface molecules. (4)

The vertebrate neural retina is made up of three distinct layers of neuronal cell bodies: the ganglion cell layer nearest the lens, intermediate layer composed of bipolar, horizontal, and amacrine cells, and the photoreceptor cell layer(5). These three distinct layers are separated by the inner and outer plexiform layer(7,5). The photoreceptor outer segment consists of tightly packed membranous light capturing disks called rhodopsin or cone opsin molecules. The central area of the retina has avascular fovea zone which is used for high-resolution central color vision. The photoreceptor cells receive their nutrients from the RPE cells located behind the retina. The RPE has tight intercellular junctions and a basement membrane called Bruch’s membrane which forms the blood-retina permeability barrier. The RPE is also involved in the uptake of vitamin A from the chorodial capillaries, the isomerization of photobleached retinoids, the phagocytosis of shed photoreceptor outer segments, and the absorption of unwanted light by melanin granules (8,9,5).

The interphotoreceptor matrix is a space between the RPE and the neural retina and it is composed of extracellular components including a variety of glycoprotiens and glycosaminoglycans that are important in maintaining the retina-RPE attachment (10). During viral transduction delivery, AAV vector is introduced into the interphotoreceptor matrix, using a subretinal injection ( 11). The fluid is then absorbed through the RPE by polarized apicalto-basal pumping process.

Photoreceptor cells and Retinal pigment epithelium (RPE) appear to be the major causative sites for retinal degenerative diseases. Majority of the genes that are associated with retinal degenerative diseases are expressed either in photoreceptor cells or RPE. The RPE provides photoreceptors with needed nutrients, growth factors, and oxygen. The photoreceptor rod cells provide nutrient factors for the cones and also produce proteins that are important in maintaining the inner retinal integrity. (12,13)

The most successful AAV vectors are those that have been optimized by AAV serotype and that can have efficient and selective promoters to deliver high, sustained levels of the therapeutic wild-type proteins to the correct retinal cell. Another way is downregulating the defective gene by AAV-mediated delivery of ribozymes or small interfering RNA (siRNA), but it only shows a temporary effect (5).

The most prominent retinal degenerative diseases occur as variety of neovascular diseases, such as proliferative diabetic retinopathy where a high levels of hyperglycemia causes widespread closure of retinal blood vessels which leads to the formation of new blood vessels and propagation of neovascularization resulting in blindness (14).

The type of vectors used in targeting photoreceptors and RPE cells play an important role in achieving a successful treatment. When looking at transduction efficiency of either AAV2 or AAV5 vectors expressing green fluorescent protein (GFP) in the RPE and Photoreceptor cells, AAV5 vectors appears to be more efficient. Pervious experiment shows that at 15 weeks post-injection, the ratio of transgene-expressing photoreceptor cells transduced with AAV5 vs AAV2 was 400:1, and the number of viral genome copies per eye was 30 times higher for type 5 (15,5). In other similar vectors such as AAV1 vectors, seem to tranduce predominantly the RPE cells, also they were 15 times more efficient than AAV2 vectors . AAV6 vectors are very similar to AAV1 vectors in which it transduces primarily the RPE cells. AAV 3 transduces retinal cells poorly but AAV4 has constant transduction of the RPE cells. (15).

The other important determinants of retinal cell type transduction are promoter sequences. For example, in rat the murine rhodopsin gene drive the expression in AAV2, GFP reporter product was found only in rat photoreceptors, not in any other retinal cell type or in the adjacent RPE after subretinal injection. On the other hand, if ubiquitously expressed immediate-early cytomegalovirus (CMV) enhancer-promoter might not be specific to certain cell types. Other more recent promoters like the CBA promoter, a fusion of the chicken-actin promoter and CMV immediate-early enhancer allows stable GFP reporter expression in both RPE and photoreceptor cells after subretinal injections(5).

The promoters of VMD2 (Vitelliform Macular Dystrophy) and RPE65 in combination with the appropriate AAV serotype can allow specific and efficient targeting of RPE cells. Sometimes modulation of transgene expression may be necessary since strong constitutive expression of a therapeutic gene in retinal tissues could be deleterious for long-term retinal function. Different methods have been utilized for the expression modulation. One way is using exogenously regulatable promoter system in AAV vectors. For example, the tetracycline-inducible expression system uses a silencer/transactivator AAV2 vector and a separate inducible doxycline-responsive coinjection.( 16,5). When induction occurs by oral doxycycline, this system shows tight regulation of gene expression in both photoreceptor and RPE cells.

One study that was done by Royal College of Surgeons (RCS) in rat model shows that a recessive mutation in a receptor tyrposine kinase gene, mertk results in a premature stop codon and impaired phagocytosis function by RPE cells. This mutation causes the accumulation of outer segment debris in the subretinal space, which causes photoreceptor cell death. The model organism with this disease received a subretinal injection of AAV serotype 2 carrying a mouse Mertk cDNA under the control of either the CMV or RPE65 promoters. This treatment was found to prolong photoreceptor cell survival for several months (17) and also the number of photoreceptor was 2.5 fold higher in AAV-Mertk- treated eyes compared with controls 9 weeks after injection, also they found decreased amount of debris in the subretinal space.

The protein RPE65 is used in the retinoid cycle where the all-trans-retinol within the rod outer segment is isomerized to its 11-cis form and oxidized to 11-cis retinal before it goes back to the photoreceptor and joins with opsin molecule to form functional rodopsin (18). In animal knockout model (RPE65-/-), gene transfer experiment shows that early intraocular delivery of human RPE65 vector on embryonic day 14 shows efficient transduction of retinal pigment epithelium in the RPE65-/- knockout mice and rescues visual functions. This shows successful gene therapy can be attributed to early intraocular deliver to the diseased animal.

Juvenile retinoschisis is a disease that affects the nerve tissue in the eye. This disease is an X-linked recessive degenerative disease of the central macula region, and it is caused by mutation in the RSI gene encoding the protein retinoschisin. Retinoschisin is produced in the photoreceptor and bipolar cells and it is critical in maintaining the synaptic integrity of the retina.(5)

Specifically the AAV 5 vector containing the wild-type human RSI cDNA driven by a mouse opsin promoter showed long-term retinal functional and structural recovery. Also the retinal structural reliability improved greatly after the treatment, characterized by an increase in the outer nuclear layer thickness( 5 )

Retinitis pigmentosa is an inherited disease which leads to progressive night blindness and loss of peripheral vision as a result of photoreceptor cell death(19,20,5). Different type of inheritance can attribute to this disease; autosomal recessive, autosomal dominant, X-linked type, etc. The main function of rhodopsin is initiating the phototransduction cascade. The opsin proteins are made in the photoreceptor inner segments and then transported to the outer segment and eventually phagocytized by the RPE cells. When mutations occur in the rhodopsin the directional protein movement is affected because the mutations can affect protein folding, stability, and intracellular trafficking. One approach is introducing AAV-delivered ribozymes designed to target and destroy a mutant mRNA(5).

The way this system operates was shown in animal model that have a mutant rhodopsin gene. The injected AAV-ribozymes were optimized in vitro and used to cleave the mutant mRNA transcript of P23H ( where most mutation occur) in vivo.

Another mutation in the rhodopsin structural protein, specifically peripherin 2 which is a membrane glycoprotein involved in the formation of photoreceptor outersegment disk, can lead to recessive RP and macular degeneration in human (19). In a mouse experiment, AAV2 carrying a wild-type peripherin 2 gene driven by a rhodopsin promoter was delivered to the mice by subretinal injection. The result showed improvement in photoreceptor structure and function which was detected by ERG (electroretinogram). The result showed improvement of photoreceptor structure and function which was detected by ERG. Also peripherin 2 was detected at the outer segment layer of the retina 2 weeks after injection and therapeutic effects were noted as soon as 3 weeks after injection. Interestingly, a well defined outer segment containing both peripherin2 and rhodopsin was present 9 month after injection. (5)

Since apoptosis can be the cause of photoreceptor death in most of the retinal dystrophies It has been known that survival factors and antiapoptoic reagents can be an alternative treatment if the mutation is unknown for gene replacement therapy. Experiments were carried out to study whether supplying AAV2 vectors with cDNA for glial cell line-derived neurotrophic factor (GDNF) can have an anti-apoptosis effect on the rod cells(21,5). In looking at an animal model, the opsin transgene contains a truncated protein lacking the last 15 amino acids of the C terminus, which causes alteration in rhodopsin transport to the outer segment and leads to retinal degeneration (5) When the AAV2-CBA-GDNF vector is administered to the subretinal space, photoreceptor stabilized and rod photoreceptors increased and this was seen in the improved function of the ERG analysis.( 21).

Ocular neovascularization(NV) is an the abnormal formation of new capillaries from already existing blood vessels in the eye, and this is a characteristics for ocular diseases such as diabetic retinopathy (DR), retinopathy of prematurity (ROP) and (wet form) age-related macular degeneration (AMD). One of the main players in these diseases is VEGF (Vascular endothelial growth factor) which is known to induce vessel leakage and which is also known to be angiogenic(5) . In normal tissues VEGF stimulates endothelial cell proliferation in a dose dependent manner, but such activity is lost with other angiogenic factors. (22). Many angiostatic factors have been shown to counteract the effect of increasing local VEGF. For example, pigment epithelium-derived factor (PEDF) acts as an inhibitor of angiogenesis. The secretion of PEDF is noticeably decreased under hypoxic conditions allowing the endothelial mitogenic activity of VEGF to dominate, suggesting that the loss of PEDF plays a central role in the development of ischemia-driven NV. One interesting clinical finding shows that the levels of PEDF in aqueous humor of human are decreased with increasing age, indicating that the reduction may lead to the development of AMD.( 23,5).

In animal model, an AAV with human PEDF cDNA under the control of the CMV promoter prevented choroidal and retinal NV.( 24). The finding suggests that PEDF expression using AAV vectors can be implemented to treat NV.( 25). One great advantage of this treatment is that the PEDF is able to diffuse through sclera tissue, making this angiostatic agent appears to be relatively independent of the intraocular site of administration. These type of therapeutic methods can be important in a clinical setting since intravitreal and periocular injections can be less invasive than subretinal injections (5).

Clinical trial was done on three young adults with RPE65-LCA. They used AAV-2 vectors for RPE65 gene replacement therapy and visual function were tested within 90 days after the intervention. The patients showed an increased in visual sensitivity at 30 days after treatment. Also in order to analyze restored visual potential, they correlated degree of light sensitivity to the levels of photoreceptors. They found that intervention helped gain the loss of light sensitivity resulting from the disease, but resensitization kinetics of the treated rods were very slow and required at least 8hr to gain full sensitivity(6).

References

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References

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