Adeno associated virus and gene therapy of the human retina
Encyclopedia
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.
Retina
l 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 effect
s.
In retinal gene therapy, the most widely used vectors for ocular gene delivery are based on adeno-associated 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.
es with a non-enveloped icosahedral capsid of approximately 22 nm.
It was discovered in 1965 as a contaminant of adenovirus (AD) preparations. 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). 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
). The rep is composed of four overlapping genes encoding rep protein which is required for the AAV life cycle. The cap gene contains sequences of capsid proteins: virion protein 1 (VP1), VP2 and VP3, which ultimately forms a capsid of icosahedral symmetry
.
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. The one side of the AAV genome contains two promoters at position 5 (p5) and 19 p(19). These two promoters produce two overlapping messenger ribonucleic acids
(mRNAs) and through the process of splicing different lengths of the (mRNAs) are produced. There are four different possibilities of mRNA products; four Rep proteins can be synthesized with different sizes: Rep78, Rep68, Rep52, and Rep40. 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.
and the process of internalization occurs with the help of co-receptors αvβ5 heterodimers, fibroblast growth factor
s receptor type 1 and the hepatocyte growth factor
receptor, c-Met.
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. 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
. 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.
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.
al cell bodies: the ganglion cell
layer nearest the lens, intermediate layer composed of bipolar
, horizontal
, and amacrine cell
s, and the photoreceptor cell layer. These three distinct layers are separated by the inner and outer plexiform layer. 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 nutrient
s 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.
The interphotoreceptor matrix is a space between the RPE and the neural retina and it is composed of extracellular components including a variety of glycoprotein
s and glycosaminoglycan
s that are important in maintaining the retina-RPE attachment. During viral transduction delivery, AAV vector is introduced into the interphotoreceptor matrix, using a subretinal injection
. 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 disease
s are expressed either in photoreceptor cells or RPE. The RPE provides photoreceptors with needed nutrients, growth factor
s, and oxygen
. The photoreceptor rod cells provide nutrient factors for the cones and produce proteins that are important in maintaining the inner retinal integrity.
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 ribozyme
s or small interfering RNA
(siRNA), but it only shows a temporary effect.
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 vessel
s which leads to the formation of new blood vessels and propagation of neovascularization
resulting in blindness.
(GFP) in the RPE and Photoreceptor cells, AAV5 vectors appears to be more efficient. Previous 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. 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.
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.
, this system shows tight regulation of gene expression in both photoreceptor and RPE cells.
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.
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.
is an inherited disease which leads to progressive night blindness and loss of peripheral vision
as a result of photoreceptor cell death. 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, 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.
and used to cleave the mutant mRNA transcript of P23H (where most mutation occur) in vivo.
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.
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 cell
s.
In looking at an animal model, the opsin transgene contains a truncated protein lacking the last 15 amino acid
s of the C terminus, which causes alteration in rhodopsin transport to the outer segment and leads to retinal degeneration 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.
. 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.
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.
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).
Gene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...
is the use of genetic material (DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
) inserted into a patient’s cell for the treatment of an inherited or acquired diseases
Acquired disorder
An acquired disorder is a medical condition which develops post-fetally; in contrast with a congenital disorder, which is present at birth. A congenital disorder may be antecedent to an acquired disorder ....
. There are many medical conditions that are a result of mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
in patient’s gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
. Gene therapy tries to introduce functional form of that gene into the patient’s cell nucleus
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
in order to compensate for the mutated gene.
Retina
Retina
The vertebrate retina is a light-sensitive tissue lining the inner surface of the eye. The optics of the eye create an image of the visual world on the retina, which serves much the same function as the film in a camera. Light striking the retina initiates a cascade of chemical and electrical...
l gene therapy holds great promise in treating different non-inherited and inherited blindness
Blindness
Blindness is the condition of lacking visual perception due to physiological or neurological factors.Various scales have been developed to describe the extent of vision loss and define blindness...
. The first gene therapy trial for inherited retinal disease took place in 2007 at Moorfield Eye Hospital and University College London
University College London
University College London is a public research university located in London, United Kingdom and the oldest and largest constituent college of the federal University of London...
’s Institute of Ophthalmology. This therapy was done to treat Leber's congenital amaurosis
Leber's congenital amaurosis
Leber's congenital amaurosis is a rare inherited eye disease that appears at birth or in the first few months of life, and affects around 1 in 80,000 of the population.It was first described by Theodor Leber in the 19th century...
diseases which is an inherited blinding disease caused by mutations in RPE65
RPE65
Retinal pigment epithelium-specific 65 kDa protein is a protein that in humans is encoded by the RPE65 gene.-Function:The retinal pigment epithelium-specific 65 kDa protein is located in the retinal pigment epithelium and is involved in the conversion of all-trans retinol to 11-cis retinal during...
gene. The result was somewhat promising, showing slight increase in vision and most importantly there were no side effect
Side effect
In medicine, a side effect is an effect, whether therapeutic or adverse, that is secondary to the one intended; although the term is predominantly employed to describe adverse effects, it can also apply to beneficial, but unintended, consequences of the use of a drug.Occasionally, drugs are...
s.
In retinal gene therapy, the most widely used vectors for ocular gene delivery are based on adeno-associated virus
Adeno-associated virus
Adeno-associated virus is a small virus which infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of...
. The great advantage in using adeno-associated virus for the gene therapy is that it poses minimal immune responses and mediates long-term transgene
Transgene
A transgene is a gene or genetic material that has been transferred naturally or by any of a number of genetic engineering techniques from one organism to another....
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.
The Human Adeno-associated Virus
The human adeno-associated virus (AAV) is one of the smallest virusVirus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
es with a non-enveloped icosahedral capsid of approximately 22 nm.
It was discovered in 1965 as a contaminant of adenovirus (AD) preparations. The AAV has a linear single-stranded DNA genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
that consists of 145 nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
-long inverted terminal repeat (ITR) and a size of approximately 4.7-kilobases (kb). Since a virus does not encode a polymerase
Polymerase
A polymerase is an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA.The primary function of a polymerase is the polymerization of new DNA or RNA against an existing DNA or RNA template in the processes of replication and transcription...
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
Capsid
A capsid is the protein shell of a virus. It consists of several oligomeric structural subunits made of protein called protomers. The observable 3-dimensional morphological subunits, which may or may not correspond to individual proteins, are called capsomeres. The capsid encloses the genetic...
). The rep is composed of four overlapping genes encoding rep protein which is required for the AAV life cycle. The cap gene contains sequences of capsid proteins: virion protein 1 (VP1), VP2 and VP3, which ultimately forms a capsid of icosahedral symmetry
Icosahedral symmetry
A regular icosahedron has 60 rotational symmetries, and a symmetry order of 120 including transformations that combine a reflection and a rotation...
.
An interesting property of the ITRs is that they can form a hairpin
Stem-loop
Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions,...
, which contributes to self-priming that allows primase
Primase
DNA primase is an enzyme involved in the replication of DNA.Primase catalyzes the synthesis of a short RNA segment called a primer complementary to a ssDNA template...
-independent synthesis of the second DNA strand and they are also required for the integration of the AAV DNA into the host cell genome. The one side of the AAV genome contains two promoters at position 5 (p5) and 19 p(19). These two promoters produce two overlapping messenger ribonucleic acids
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
(mRNAs) and through the process of splicing different lengths of the (mRNAs) are produced. There are four different possibilities of mRNA products; four Rep proteins can be synthesized with different sizes: Rep78, Rep68, Rep52, and Rep40. 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.
Host infection
The primary attachment site for AAV2 virions is the heparin sulfate proteoglycanProteoglycan
Proteoglycans are proteins that are heavily glycosylated. The basic proteoglycan unit consists of a "core protein" with one or more covalently attached glycosaminoglycan chain. The point of attachment is a Ser residue to which the glycosaminoglycan is joined through a tetrasaccharide bridge...
and the process of internalization occurs with the help of co-receptors αvβ5 heterodimers, fibroblast growth factor
Fibroblast growth factor
Fibroblast growth factors, or FGFs, are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. The FGFs are heparin-binding proteins and interactions with cell-surface associated heparan sulfate proteoglycans have been shown to be essential for FGF signal...
s receptor type 1 and the hepatocyte growth factor
Hepatocyte growth factor
Hepatocyte growth factor/scatter factor is a paracrine cellular growth, motility and morphogenic factor. It is secreted by mesenchymal cells and targets and acts primarily upon epithelial cells and endothelial cells, but also acts on haemopoietic progenitor cells...
receptor, c-Met.
Once the AAV has entered the host cell, it can go through two different pathways of life cycle
Biological life cycle
A life cycle is a period involving all different generations of a species succeeding each other through means of reproduction, whether through asexual reproduction or sexual reproduction...
: the lytic
Lytic cycle
The lytic cycle is one of the two cycles of viral reproduction, the other being the lysogenic cycle. The lytic cycle is typically considered the main method of viral replication, since it results in the destruction of the infected cell...
or the lysogenic. The lytic stage occurs in cells infected with a helper virus
Helper virus
A helper virus is a virus used when producing copies of a helper dependent viral vector which does not have the ability to replicate on its own. The helper virus is used to coinfect cells alongside the viral vector and provides the necessary enzymes for replication of the genome of the viral vector....
such as Adeno virus (AD) or herpes simplex virus
Herpes simplex virus
Herpes simplex virus 1 and 2 , also known as Human herpes virus 1 and 2 , are two members of the herpes virus family, Herpesviridae, that infect humans. Both HSV-1 and HSV-2 are ubiquitous and contagious...
(HSV), whereas the lysogenic stage is established in host cells in the absence of (or, without) helper virus. During AAV infection of just a human cell, its gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
program is auto-repressed and the virus genome integrates into a region of roughly 2-kb on the long arm of human chromosome 19
Chromosome 19 (human)
125px|rightChromosome 19 is one of the 23 pairs of chromosomes in humans. People normally have two copies of this chromosome. Chromosome 19 spans more than 63 million base pairs and represents between 2 and 2.5 percent of the total DNA in cells.Identifying genes on each chromosome is an active...
. 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.
Tropism modification
One important factor in gene deliveryGene delivery
Gene delivery is the process of introducing foreign DNA into host cells. Gene delivery is, for example, one of the steps necessary for gene therapy and the genetic modification of crops. There are many different methods of gene delivery developed for a various types of cells and tissues, from...
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.
Important physiological components in retinal gene therapy
The vertebrate neural retina is made up of three distinct layers of neuronNeuron
A neuron is an electrically excitable cell that processes and transmits information by electrical and chemical signaling. Chemical signaling occurs via synapses, specialized connections with other cells. Neurons connect to each other to form networks. Neurons are the core components of the nervous...
al cell bodies: the ganglion cell
Ganglion cell
A retinal ganglion cell is a type of neuron located near the inner surface of the retina of the eye. It receives visual information from photoreceptors via two intermediate neuron types: bipolar cells and amacrine cells...
layer nearest the lens, intermediate layer composed of bipolar
Bipolar cell
As a part of the retina, the bipolar cell exists between photoreceptors and ganglion cells. They act, directly or indirectly, to transmit signals from the photoreceptors to the ganglion cells.-Overview:...
, horizontal
Horizontal cell
Horizontal cells are the laterally interconnecting neurons in the outer plexiform layer of the retina of mammalian eyes. They help integrate and regulate the input from multiple photoreceptor cells...
, and amacrine cell
Amacrine cell
Amacrine cells are interneurons in the retina. Amacrine cells are responsible for 70% of input to retinal ganglion cells. Bipolar cells, which are responsible for the other 30% of input to retinal ganglia, are regulated by amacrine cells.-Overview:...
s, and the photoreceptor cell layer. These three distinct layers are separated by the inner and outer plexiform layer. The photoreceptor outer segment consists of tightly packed membranous light capturing disks called rhodopsin
Rhodopsin
Rhodopsin, also known as visual purple, is a biological pigment of the retina that is responsible for both the formation of the photoreceptor cells and the first events in the perception of light. Rhodopsins belong to the G-protein coupled receptor family and are extremely sensitive to light,...
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 nutrient
Nutrient
A nutrient is a chemical that an organism needs to live and grow or a substance used in an organism's metabolism which must be taken in from its environment. They are used to build and repair tissues, regulate body processes and are converted to and used as energy...
s from the RPE cells located behind the retina. The RPE has tight intercellular junctions and a basement membrane called Bruch's membrane
Bruch's membrane
Bruch's membrane is the innermost layer of the choroid. It is also called the vitreous lamina, because of its glassy microscopic appearance.It is 2–4 μm thick.-Layers:Bruch's membrane consists of five layers :...
which forms the blood-retina permeability barrier. The RPE is also involved in the uptake of vitamin A
Vitamin A
Vitamin A is a vitamin that is needed by the retina of the eye in the form of a specific metabolite, the light-absorbing molecule retinal, that is necessary for both low-light and color vision...
from the chorodial capillaries, the isomerization of photobleached retinoids, the phagocytosis of shed photoreceptor outer segments, and the absorption of unwanted light by melanin
Melanin
Melanin is a pigment that is ubiquitous in nature, being found in most organisms . In animals melanin pigments are derivatives of the amino acid tyrosine. The most common form of biological melanin is eumelanin, a brown-black polymer of dihydroxyindole carboxylic acids, and their reduced forms...
granules.
The interphotoreceptor matrix is a space between the RPE and the neural retina and it is composed of extracellular components including a variety of glycoprotein
Glycoprotein
Glycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending...
s and glycosaminoglycan
Glycosaminoglycan
Glycosaminoglycans or mucopolysaccharides are long unbranched polysaccharides consisting of a repeating disaccharide unit. The repeating unit consists of a hexose or a hexuronic acid, linked to a hexosamine .-Production:Protein cores made in the rough endoplasmic reticulum are posttranslationally...
s that are important in maintaining the retina-RPE attachment. During viral transduction delivery, AAV vector is introduced into the interphotoreceptor matrix, using a subretinal injection
Injection (medicine)
An injection is an infusion method of putting fluid into the body, usually with a hollow needle and a syringe which is pierced through the skin to a sufficient depth for the material to be forced into the body...
. 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 disease
Degenerative disease
A degenerative disease, also called neurodegenerative disease, is a disease in which the function or structure of the affected tissues or organs will progressively deteriorate over time, whether due to normal bodily wear or lifestyle choices such as exercise or eating habits...
s are expressed either in photoreceptor cells or RPE. The RPE provides photoreceptors with needed nutrients, growth factor
Growth factor
A growth factor is a naturally occurring substance capable of stimulating cellular growth, proliferation and cellular differentiation. Usually it is a protein or a steroid hormone. Growth factors are important for regulating a variety of cellular processes....
s, and oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
. The photoreceptor rod cells provide nutrient factors for the cones and produce proteins that are important in maintaining the inner retinal integrity.
The most successful AAV vectors are those that have been optimized by AAV serotype
Serotype
Serotype or serovar refers to distinct variations within a subspecies of bacteria or viruses. These microorganisms, viruses, or cells are classified together based on their cell surface antigens...
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 ribozyme
Ribozyme
A ribozyme is an RNA molecule with a well defined tertiary structure that enables it to catalyze a chemical reaction. Ribozyme means ribonucleic acid enzyme. It may also be called an RNA enzyme or catalytic RNA. Many natural ribozymes catalyze either the hydrolysis of one of their own...
s or small interfering RNA
Small interfering RNA
Small interfering RNA , sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology. The most notable role of siRNA is its involvement in the RNA interference pathway, where it...
(siRNA), but it only shows a temporary effect.
The most prominent retinal degenerative diseases occur as variety of neovascular diseases, such as proliferative diabetic retinopathy
Diabetic retinopathy
Diabetic retinopathy is retinopathy caused by complications of diabetes mellitus, which can eventually lead to blindness....
where a high levels of hyperglycemia
Hyperglycemia
Hyperglycemia or Hyperglycæmia, or high blood sugar, is a condition in which an excessive amount of glucose circulates in the blood plasma. This is generally a glucose level higher than 13.5mmol/l , but symptoms may not start to become noticeable until even higher values such as 15-20 mmol/l...
causes widespread closure of retinal blood vessel
Blood vessel
The blood vessels are the part of the circulatory system that transports blood throughout the body. There are three major types of blood vessels: the arteries, which carry the blood away from the heart; the capillaries, which enable the actual exchange of water and chemicals between the blood and...
s which leads to the formation of new blood vessels and propagation of neovascularization
Neovascularization
Neovascularization is the formation of functional microvascular networks with red blood cell perfusion. Neovascularization differs from angiogenesis in that angiogenesis is mainly characterized by the protrusion and outgrowth of capillary buds and sprouts from pre-existing blood vessels.In...
resulting in blindness.
AAV Targeting of photoreceptors and RPE cells
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 proteinGreen fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
(GFP) in the RPE and Photoreceptor cells, AAV5 vectors appears to be more efficient. Previous 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. In other similar vectors
Viral vector
Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism or in cell culture . Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect...
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.
Promoter sequence
The other important determinants of retinal cell type transduction are promoter sequences. For example, in ratRat
Rats are various medium-sized, long-tailed rodents of the superfamily Muroidea. "True rats" are members of the genus Rattus, the most important of which to humans are the black rat, Rattus rattus, and the brown rat, Rattus norvegicus...
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
Cytomegalovirus
Cytomegalovirus is a viral genus of the viral group known as Herpesviridae or herpesviruses. It is typically abbreviated as CMV: The species that infects humans is commonly known as human CMV or human herpesvirus-5 , and is the most studied of all cytomegaloviruses...
(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.
Modulation of expression
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. When induction occurs by oral doxycyclineDoxycycline
Doxycycline INN is a member of the tetracycline antibiotics group, and is commonly used to treat a variety of infections. Doxycycline is a semisynthetic tetracycline invented and clinically developed in the early 1960s by Pfizer Inc. and marketed under the brand name Vibramycin. Vibramycin...
, this system shows tight regulation of gene expression in both photoreceptor and RPE cells.
Animal model study 1
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 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.Animal model study 2
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. 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.Targeting the photoreceptor cells
Juvenile retinoschisisRetinoschisis
Retinoschisis is an eye disease characterized by the abnormal splitting of the retina's neurosensory layers, usually in the outer plexiform layer, resulting in a loss of vision in the corresponding visual field in some rarer forms...
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.
Animal model study 1
Specifically the AAV 5 vector containing the wild-type human RSI cDNA driven by a mouseMouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...
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
Outer nuclear layer
The outer nuclear layer , is one of the layers of the vertebrate retina, the light-detecting portion of the eye...
thickness.
Retinitis pigmentosa
Retinitis pigmentosaRetinitis pigmentosa
Retinitis pigmentosa is a group of genetic eye conditions that leads to incurable blindness. In the progression of symptoms for RP, night blindness generally precedes tunnel vision by years or even decades. Many people with RP do not become legally blind until their 40s or 50s and retain some...
is an inherited disease which leads to progressive night blindness and loss of peripheral vision
Peripheral vision
Peripheral vision is a part of vision that occurs outside the very center of gaze. There is a broad set of non-central points in the field of view that is included in the notion of peripheral vision...
as a result of photoreceptor cell death. 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, 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
Protein folding
Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....
, stability, and intracellular trafficking. One approach is introducing AAV-delivered ribozymes designed to target and destroy a mutant mRNA.
Animal model study: 1
The way this system operates was shown in animal model that have a mutant rhodopsin gene. The injected AAV-ribozymes were optimized in vitroIn vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
and used to cleave the mutant mRNA transcript of P23H (where most mutation occur) in vivo.
Animal model study: 2
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 degenerationMacular degeneration
Age-related macular degeneration is a medical condition which usually affects older adults and results in a loss of vision in the center of the visual field because of damage to the retina. It occurs in “dry” and “wet” forms. It is a major cause of blindness and visual impairment in older adults...
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.
Animal model study: 3
Since apoptosisApoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
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
Glial cell
Glial cells, sometimes called neuroglia or simply glia , are non-neuronal cells that maintain homeostasis, form myelin, and provide support and protection for neurons in the brain, and for neurons in other parts of the nervous system such as in the autonomous nervous system...
line-derived neurotrophic factor (GDNF) can have an anti-apoptosis effect on the rod cell
Rod cell
Rod cells, or rods, are photoreceptor cells in the retina of the eye that can function in less intense light than can the other type of visual photoreceptor, cone cells. Named for their cylindrical shape, rods are concentrated at the outer edges of the retina and are used in peripheral vision. On...
s.
In looking at an animal model, the opsin transgene contains a truncated protein lacking the last 15 amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s of the C terminus, which causes alteration in rhodopsin transport to the outer segment and leads to retinal degeneration 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.
Treatment for Neovascular Retinal Disease
Ocular neovascularization (NV) is 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. In normal tissues VEGF stimulates endothelial cell proliferation in a dose dependent manner, but such activity is lost with other angiogenic factors. 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 angiogenesisAngiogenesis
Angiogenesis is the physiological process involving the growth of new blood vessels from pre-existing vessels. Though there has been some debate over terminology, vasculogenesis is the term used for spontaneous blood-vessel formation, and intussusception is the term for the formation of new blood...
. 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
Ischemia
In medicine, ischemia is a restriction in blood supply, generally due to factors in the blood vessels, with resultant damage or dysfunction of tissue. It may also be spelled ischaemia or ischæmia...
-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.
Animal model study 1
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 scleraSclera
The sclera , also known as the white or white of the eye, is the opaque , fibrous, protective, outer layer of the eye containing collagen and elastic fiber. In the development of the embryo, the sclera is derived from the neural crest...
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.
Patient related Clinical trial
A clinical trialClinical trial
Clinical trials are a set of procedures in medical research and drug development that are conducted to allow safety and efficacy data to be collected for health interventions...
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
Light sensitivity
Light sensitivity or photosensitivity is an increase in the reactivity of the skin to sunlight. Apart from vision, human beings have many physiological and psychological responses to light. In rare individuals an atypical response may result in serious discomfort, disease, or injury. Some drugs...
resulting from the disease, but resensitization kinetics of the treated rods were very slow and required at least 8hr to gain full sensitivity(6).