glaucoma || gene therapy in glaucoma

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657 64 Gene Therapy in Glaucoma STUART J MCKINNON enzymes, and therefore invade a host cell to obtain access to these functions. Rather than dividing as most microor- ganisms do, viruses reproduce in host cells by assembling subunits into infectious particles, consisting of DNA or RNA surrounded by a symmetric protein coat (capsid). The capsid protects the internal nucleic acids from the external environment, confers antigenicity, and mediates attach- ment to susceptible cells. The viral life cycle consists of two phases, the intracellular phase in which the viral nucleic acids are replicated and packaged within the capsid to form the virion, and the infectious extracellular phase in which the virion invades the host causing cellular alteration or death. Viruses are classified according to the type of nucleic acid (DNA or RNA), the secondary structure of the nucleic acid (single-stranded, double-stranded, circular or coiled), symmetry of the capsid (icosahedral or helical), and the presence of a cell-derived envelope (naked or enveloped). 2 We will limit this review to the common viral vector types that have been employed in ocular gene therapy, specifically adenovirus, adeno-associated virus (AAV), and retroviruses such as lentivirus. Adenovirus One of the first viruses to be employed as a vector for gene transfer is adenovirus, which in its native form causes res- piratory tract and ocular infections in humans. Adenovirus has been well characterized as to genetic makeup, gene functions and interactions with the infected host cell. Ade- novirus consists of a 36-kilobase (kb) linear double-stranded DNA genome surrounded by a non-enveloped (naked) icosahedral capsid. Adsorption of adenovirus to the host cell involves binding of fiber proteins that project from each of the 20 vertices of the icosahedral capsid to a coxsackie- virus adenovirus receptor (CAR). 3 This receptor is present in a wide range of cell types, but when absent leads to viral toxicity and resistance to transgene expression. Recently, several CAR-independent adenovirus vectors that incorpo- rate different capsid proteins have been generated to improve adenoviral vector transduction. Restricting viral tropism to selected cell types has also been furthered by altering viral genomes to produce virions with modified capsids (pseudotyping). 4 The first generation of adenoviral vectors was engineered to render the virus replication defective, by deleting the E1A, E1B and E3 genes. E1A proteins are the first viral proteins to be expressed after transduction, and activate transcription by modifying transcription factors and tran- scriptional regulators. E1A proteins also interact with the retinoblastoma protein (pRB) to induce quiescent cells to enter the S phase of the cell cycle. 5 The E1B protein is required for efficient accumulation of viral messenger RNA Introduction Genetic manipulation of the mammalian central nervous system has progressed rapidly over the past two decades. Gene transfer has been a well-characterized technique in molecular and cellular biology for many years, and the ability to express a protein in mammalian cell culture has given us a great deal of information concerning both normal and pathological cellular processes. The use of transgenic animals has extended this approach, but the use of these animals is time-consuming and subject to difficul- ties in interpretation. To allow more controlled genetic manipulations, neurobiologists have taken advantage of pathogenic viruses to develop systems to deliver genes of interest (transgenes) to specific neuronal cell populations. For glaucoma, targets of gene therapy approaches include aqueous humor outflow modification in the trabecular meshwork (TM), and neuroprotection of retinal ganglion cells (RGCs) and the optic nerve. Virus Classification Viruses are small, infectious, intracellular parasites char- acterized by their simple organization, mode of replica- tion and nucleic acid composition. Viruses intrinsically lack the ability to produce energy or synthesize proteins and Summary Glaucoma is uniquely well-suited to gene therapy. The trabecular meshwork or Schlemm’s canal can be targeted by anterior chamber injection due to aqueous humor outflow, and RGCs can be targeted with intravitreal injection due to their proximal inner retinal location to the vitreous. Although these efforts are still in their relative infancy, successful gene therapy experiments have been directed toward modification of trabecular meshwork extracellular matrix and cytoskeleton components using adenovirus and lentivirus constructs, and modulation of apoptosis and intracellular signaling pathways in RGCs has been achieved with AAV constructs. However, idiosyncratic immune responses leading to human deaths have been reported using systemic administration of both adenoviral and AAV vectors. The decreased amount of viral vector used for ocular administration should reduce this risk considerably, but caution is certainly warranted with any human gene therapy that is undertaken. Nonviral techniques are also being employed for gene transfer, using cationic lipid delivery systems (lipofection) or the use of electric current (electroporation) to deliver DNA sequences into target cells. These nonviral techniques provide a complementary approach that could minimize the immune responses that can limit therapeutic viral vector gene transfer trials. 1

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Page 1: Glaucoma || Gene Therapy in Glaucoma

657

64  Gene Therapy in GlaucomaSTUART J MCKINNON

enzymes, and therefore invade a host cell to obtain access to these functions. Rather than dividing as most microor-ganisms do, viruses reproduce in host cells by assembling subunits into infectious particles, consisting of DNA or RNA surrounded by a symmetric protein coat (capsid). The capsid protects the internal nucleic acids from the external environment, confers antigenicity, and mediates attach-ment to susceptible cells. The viral life cycle consists of two phases, the intracellular phase in which the viral nucleic acids are replicated and packaged within the capsid to form the virion, and the infectious extracellular phase in which the virion invades the host causing cellular alteration or death. Viruses are classified according to the type of nucleic acid (DNA or RNA), the secondary structure of the nucleic acid (single-stranded, double-stranded, circular or coiled), symmetry of the capsid (icosahedral or helical), and the presence of a cell-derived envelope (naked or enveloped).2 We will limit this review to the common viral vector types that have been employed in ocular gene therapy, specifically adenovirus, adeno-associated virus (AAV), and retroviruses such as lentivirus.

Adenovirus

One of the first viruses to be employed as a vector for gene transfer is adenovirus, which in its native form causes res-piratory tract and ocular infections in humans. Adenovirus has been well characterized as to genetic makeup, gene functions and interactions with the infected host cell. Ade-novirus consists of a 36-kilobase (kb) linear double-stranded DNA genome surrounded by a non-enveloped (naked) icosahedral capsid. Adsorption of adenovirus to the host cell involves binding of fiber proteins that project from each of the 20 vertices of the icosahedral capsid to a coxsackie-virus adenovirus receptor (CAR).3 This receptor is present in a wide range of cell types, but when absent leads to viral toxicity and resistance to transgene expression. Recently, several CAR-independent adenovirus vectors that incorpo-rate different capsid proteins have been generated to improve adenoviral vector transduction. Restricting viral tropism to selected cell types has also been furthered by altering viral genomes to produce virions with modified capsids (pseudotyping).4

The first generation of adenoviral vectors was engineered to render the virus replication defective, by deleting the E1A, E1B and E3 genes. E1A proteins are the first viral proteins to be expressed after transduction, and activate transcription by modifying transcription factors and tran-scriptional regulators. E1A proteins also interact with the retinoblastoma protein (pRB) to induce quiescent cells to enter the S phase of the cell cycle.5 The E1B protein is required for efficient accumulation of viral messenger RNA

Introduction

Genetic manipulation of the mammalian central nervous system has progressed rapidly over the past two decades. Gene transfer has been a well-characterized technique in molecular and cellular biology for many years, and the ability to express a protein in mammalian cell culture has given us a great deal of information concerning both normal and pathological cellular processes. The use of transgenic animals has extended this approach, but the use of these animals is time-consuming and subject to difficul-ties in interpretation. To allow more controlled genetic manipulations, neurobiologists have taken advantage of pathogenic viruses to develop systems to deliver genes of interest (transgenes) to specific neuronal cell populations. For glaucoma, targets of gene therapy approaches include aqueous humor outflow modification in the trabecular meshwork (TM), and neuroprotection of retinal ganglion cells (RGCs) and the optic nerve.

Virus Classification

Viruses are small, infectious, intracellular parasites char-acterized by their simple organization, mode of replica-tion and nucleic acid composition. Viruses intrinsically lack the ability to produce energy or synthesize proteins and

Summary

Glaucoma is uniquely well-suited to gene therapy. The trabecular meshwork or Schlemm’s canal can be targeted by anterior chamber injection due to aqueous humor outflow, and RGCs can be targeted with intravitreal injection due to their proximal inner retinal location to the vitreous. Although these efforts are still in their relative infancy, successful gene therapy experiments have been directed toward modification of trabecular meshwork extracellular matrix and cytoskeleton components using adenovirus and lentivirus constructs, and modulation of apoptosis and intracellular signaling pathways in RGCs has been achieved with AAV constructs. However, idiosyncratic immune responses leading to human deaths have been reported using systemic administration of both adenoviral and AAV vectors. The decreased amount of viral vector used for ocular administration should reduce this risk considerably, but caution is certainly warranted with any human gene therapy that is undertaken. Nonviral techniques are also being employed for gene transfer, using cationic lipid delivery systems (lipofection) or the use of electric current (electroporation) to deliver DNA sequences into target cells. These nonviral techniques provide a complementary approach that could minimize the immune responses that can limit therapeutic viral vector gene transfer trials.1

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SECTION 8 • New Horizons 658

seropositive for AAV. Wild-type (wt) AAV requires a helper virus (adenovirus, herpesvirus, or vaccinia virus) to estab-lish infectivity. In the absence of helper virus or toxic chal-lenge, wtAAV integrates into chromosome 19 of the human genome. Recombinant AAV (rAAV) vectors have 96% of the viral genome removed, leaving only the two 145 base-pair ITRs. Deleted portions of the AAV genome code for structural capsid (Cap) proteins and nonstructural replica-tion (Rep) proteins (Fig. 64-2). The advantage of AAV for use as vectors lies in the fact that the absence of Rep and Cap viral sequences means that no viral protein synthesis occurs following transduction, minimizing the amount of foreign protein available to trigger immune responses. Therefore, rAAV vectors are considered to have one of the highest biosafety ratings among all viral vectors. Another beneficial feature of AAV for gene transfer is its ability to infect both dividing and non-dividing cells.10 Due to the small size of the AAV genome, a limitation exists as to the amount of DNA that can be inserted, to less than 5 kb.11 Because rAAV vectors lack the Rep sequences responsible for integration into chromosomal DNA, the AAV genome

Figure 64-1 Adenoviral vector structure. (A) Ad-GFP incorporating IRES expressing human recombinant GFP. (B) Ad-C3-GFP, which has the C3 gene cloned into the multiple cloning site of the backbone, expresses C3 transferase and human recombinant GFP. LITR: left inverted terminal repeat; MCS: multiple cloning site; IRES: internal ribosome entry site; hrGFP: human recombinant green fluorescent protein; RITR: right inverted terminal repeat; C3: C3 transferase. (Redrawn from Liu X, Hu Y, Filla MS, et al. The effect of C3 transgene expression on actin and cellular adhesions in cultured human trabecular meshwork cells and on outflow facility in organ cultured monkey eyes. Mol Vis, 2005; 11:1112–21.)

A

PromoterLITR RITRMCS IRES hrGFP Adenoviral DNA

3X HA SV40 poly AEncapsidation

B

PromoterLITR RITRC3 IRES hrGFP Adenoviral DNA

3X HA SV40 poly AEncapsidation

Figure 64-2 Map of the AAV-CBA-BDNF-WPRE virus. f1(+) origin: f1 bacteriophage origin of replication; TR: terminal repeats; CMV i.e. enhancer: cytomegalovirus immediate early enhancer; ratBDNFmyc: myc-tagged rat brain-derived neurotrophic factor sequence; WPRE: woodchuck hepatitis post-transcriptional regulatory element; bGH poly(A): bovine growth hormone polyA sequence; ColE1 ori: Escherichia coli origin of replication; ApR: ampicillin resistance sequence. (Redrawn from Martin KR, Quigley HA, Zack DJ, et al. Gene therapy with brain-derived neurotrophic factor as a protection: retinal ganglion cells in a rat glaucoma model. Invest Ophthalmol Vis Sci, 2003; 44:4357–65.)

CMV i.e. enhancer

CBA promoter

Exon 1

Intron

ratBDNFmyc

WPRE

bGH poly(A)

TR

ColE1 ori

ApR

f1(+) origin TR

CBA-BDNF-WPRE6594 bp

(mRNA), and is also expressed early. E1B modulates cell cycle progression by targeting p53, a DNA-binding tumor-suppressor protein.6 Both E1A and E1B proteins also block apoptosis, and prevent the host cell from activating intrinsic cell death machinery before virion replication can occur. E3 proteins protect the infected cell from host immune responses by preventing lysis from cytotoxic T lymphocytes and tumor necrosis factor.7 These first-generation adenovi-rus vectors caused inflammation due to low-level expres-sion of viral genes, because viral transcription still occurs despite the absence of E1A and E1B genes.3 Further genera-tions of adenovirus have incorporated a mutated E2A gene, which is required for adenoviral DNA replication, and have demonstrated reduced cytotoxic T lymphocyte infiltration.8 Despite these improvements, a human gene therapy trial involving adenovirus vectors to treat ornithine transcar-bamylase deficiency resulted in the death of 18-year-old Jesse Gelsinger from a severe immune system reaction and multiple organ system failure. As a result, the FDA sus-pended human gene therapy experiments at the University of Pennsylvania in 2000.

Prior to 1996, recombinant (E1A, E1B, E3-deleted) ade-novirus vectors were generated by homologous recombina-tion of plasmids and digested adenoviral DNA in human embryonic kidney cells (HEK 293). Vector clones were iso-lated by amplifying individual plaques, a time-consuming process. This method has been replaced by engineering a recombinant plasmid containing the DNA transgene of interest, featuring an adenoviral inverted terminal repeat (ITR) sequence that is sufficient for packaging and integra-tion, a multiple cloning site (MCS) and adenoviral E2B sequence (Fig. 64-1). The adenoviral vectors are then gen-erated by homologous recombination of plasmid in E. coli strains, followed by isolation and purification of the recom-binant adenovirus.

Adeno-Associated Virus (AAV)

Adeno-associated viruses were first noticed in the late 1960s as a contaminant of adenovirus stocks.9 AAV is a small, helper-dependent parvovirus consisting of a single-stranded 4.7-kb DNA genome surrounded by a simple, naked icosahedral capsid. The AAV vector is attractive for use in gene therapy because it is efficient, long-lived, and non-toxic. No human pathology has been reported from AAV, although approximately 85% of adult humans are

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of replication. Specific viral coat proteins, encoded by the viral env gene, enable the retrovirus to interact with a recep-tor on the host cell membrane.17 Once inside the cell cyto-plasm, uncoating of the virion occurs. A unique retroviral RNA-dependent DNA polymerase, reverse transcriptase, encoded by the pol gene, transcribes a double-stranded DNA complementary to the viral RNA, and the complementary DNA moves into the nucleus where it is incorporated into the host cell’s chromosome.18 The integrated DNA is then used as a template for transcription of retroviral RNA and translation into required viral proteins.2 The newly synthe-sized viral proteins gag (capsid), pol (reverse transcriptase) and env (envelope glycoprotein) associate with viral genomic RNA via the export and packaging sequences, rev and psi. The packaged proteins and RNA then interact with the host cell membrane resulting in encapsulation and release by budding from the cell surface of mature infec-tious virions (Fig. 64-3).19

Tumor-producing retroviruses such as Maloney murine leukemia virus achieve stable integration into the host cell genome, but only in dividing cells. Lentiviruses such as human immunodeficiency virus (HIV) and feline immuno-deficiency virus (FIV) offer the advantage of stable incorpo-ration into both dividing and nondividing cell genomes, making them excellent vectors for use in gene therapy. Lentiviruses are non-oncogenic retroviruses that produce multi-organ diseases characterized by long incubation periods and persistent infection. Five serotypes have been discovered, characterized by the mammalian hosts with which they are associated. It is imperative that the use of lentiviral vectors such as HIV for gene therapy does not cause reconstitution of a replication-competent retrovirus. To eliminate this possibility, lentiviral vectors are produced by transient cotransfection of separate plasmids that express the lentiviral transfer genome containing the transgene of interest, the lentiviral structural components gag, pol and rev, and a heterologous env protein that confers stability and broad cell tropism. Another significant modification to the transfer vector construct has been the deletion in the long terminal repeat (LTR) region, rendering the LTR tran-scriptionally inactive. These self-inactivating (SIN) vectors increase biosafety by decreasing insertional mutagenesis due to transcription from the 3′ LTR of the integrated pro-virus, and by decreased production of vector transcripts that contain the packaging signal psi.20

exists autonomously in the cytoplasm in an episomal form12 and has demonstrated stable transgene expression for over one year.13

Currently, ten different AAV serotypes (AAV-1 through AAV-10) have been classified according to differences in Cap gene sequences, conferring varying host cell binding and tropism behaviors.14 Pseudotyping has also been employed, generating hybrid rAAV vectors that contain the genome of one serotype (typically AAV-2) packaged into the capsid of another serotype, improving cellular tropism and the onset and the intensity of gene expression.15 As mentioned above, AAV depends on cotransfection with a helper virus for efficient replication. Recombinant AAV vectors have the Rep and Cap sequences replaced by the transgene of inter-est, so replication and packaging of AAV relies on similar sequences derived from helper viruses such as the adenovi-rus sequences E1A, E1B, E2A, and E4.

In the past, rAAV was produced by calcium phosphate cotransfection of adenovirus-infected 293 cells with a rAAV vector plasmid and a wild-type AAV helper plasmid. The rAAV was then purified by stepwise precipitation of rAAV with ammonium sulfate, followed by two or three rounds of CsCl density gradient centrifugation. Each gradient required fractionation and identification of the virus-containing regions by dot-blot hybridization or by polymerase chain reaction (PCR) analysis. This required up to 2 weeks for completion, and often resulted in poor recovery and poor-quality virus.16 Because of the poten-tial for contamination with unwanted adenovirus, ‘mini-adenovirus’ helper plasmids have been engineered to contain only the adenoviral genes necessary for helper function and by omitting the structural and replication adenoviral genes. Further refinements in the purification process employ heparin sulfate affinity chromatography columns that improve rAAV binding, and nonionic iodixa-nol step gradient purification, which allows more rapid and efficient separation of rAAV. Virus stocks are titrated by a quantitative PCR assay and typically yield an average of 1–4 × 1013 viral particles.11,16

Lentivirus

Retroviruses are RNA-containing viruses that are known to infect a wide variety of species, and use a unique method

Figure 64-3 Retroviral vector production. A retroviral con-struct is introduced into a packaging cell, producing RNA coding for the foreign transgene of interest. Separate viral constructs produce the retroviral structural and enzymatic proteins gag, pol and env. Foreign transgene RNA is pack-aged, encapsidated and released. The resulting retrovirus vector does not express viral packaging proteins, and the vector will not be replicated further in infected target cells. (Redrawn from Buchschacher GL Jr, Wong-Staal F. Develop-ment of lentiviral vectors for gene therapy for human diseases. Blood 2000; 95:2499–504).

Vector construct

Vector RNAVector RNA

Foreign gene

product made

Vector virus

release

Vector RNAencapsidation

Viral proteinproduction

Packaging cell Target cell

gag-pol RNA

env RNA

Foreign gene

gag

env

pol

Vector RNA

Foreign gene

product made

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two proteins (bicistronic expression), by incorporating an internal ribosome entry site (IRES) element that provides an additional ribosomal translation site. In this way, a second protein such as the reporter proteins green fluorescent protein (GFP) or lacZ can be coexpressed from the same viral vector. Efficiency of viral vector transduction can be assessed when reporter protein expression is noted in cells due to the IRES-dependent mechanism, therefore implying that the same cell is also expressing the transgene of inter-est due to the cap-dependent mechanism.31

In contrast to gene therapy techniques that are designed to enhance protein expression, it is sometimes necessary to temporally regulate protein expression or to prevent toxicity from overexpression. Such inducible gene expression systems take advantage of the tetracycline or doxycycline antibiotics, which are small lipophilic drugs that enter eukaryotic cells by passive diffusion. They are attractive as gene expression modulators because they are routinely used in both human and veterinary medicine with negligi-ble side effects.32 In a typical ‘Tet-Off ’ application, expres-sion of two vectors is necessary. The first vector expresses a tetracycline-controlled transactivator (tTA). The second vector expresses a tetracycline-response element (TRE) that activates transcription of a transgene under the control of the TRE. Upon doxycycline treatment, the target gene is conditionally turned off because tTA bound with doxycy-cline cannot bind to the TRE, and transcription is repressed.33 ‘Tet-On’ systems can also be designed where genes are con-ditionally turned on by administration of doxycycline, but these are limited by the need for use of much higher levels of doxycycline.32

Downregulation of Gene Expression Using Antisense Oligonucleotides and siRNA

Antisense therapy involves downregulation of gene expres-sion by complementary oligonucleotide binding to target mRNA. Antisense oligonucleotides are short single-stranded DNA sequences engineered to be complementary to the spe-cific ‘sense’ (5’ to 3’) orientation of mRNA coding for the targeted protein. After introduction into a host cell, the antisense oligo hybridizes to the complementary mRNA sequence, forming a heteroduplex. This causes mRNA deg-radation by RNase H, an endoribonuclease that hydrolyzes phosphodiester bonds of RNA hybridized to DNA. Antisense oligos offer the benefit of rapid manufacture, on the order of days to weeks. However, antisense oligos made from native nucleotides are prone to degradation from endog-enous nucleases. To increase resistance to degradation, syn-thetic oligos can be designed that commonly modify the ribose-phosphate backbone. A sulfur atom can be substi-tuted for oxygen in the bridging phosphate group in the nucleotide linkages of the synthesized chain. This provides a phosphorothioate oligo backbone that prevents ubiqui-tous nuclease degradation while still allowing RNase H deg-radation.34 A nitrogen atom can be substituted for oxygen at the 3’ position in the bridging phosphate group, provid-ing a phosphoramidate oligo with increased target affinity and nuclease stability.35 Morpholinos are antisense oligos with modified backbones, with nucleic acid bases bound to

Modulation of Viral Vector Expression

In order to initiate transcription, a DNA sequence exists upstream of the gene of interest to which the enzyme RNA polymerase binds. In viral vectors, these ‘promoter’ sequences have been incorporated to insure efficiency of gene expression in specific cell populations. The most common promoter is derived from cytomegalovirus (CMV), and drives expression in multiple neuronal and vascular cell types. The CMV promoter is small in size (700 bp), which makes it ideal for use in size-limited vectors such as rAAV. In ocular applications, the CMV promoter is efficient in driving expression in TM cells, but is relatively inefficient in driving expression in RGCs.21 Another commonly used pro-moter is the CMV enhancer/chicken β-actin (CBA) pro-moter. The CBA promoter enables high levels of long-term rAAV-mediated gene expression in neurons,22 and particu-larly in RGCs.23 Neuronal promoters such as human platelet-derived growth factor (PDGF) and neuron-specific enolase (NSE), and glia-specific promoters such as glial fibrillary acidic protein (GFAP) have promise for use in ocular gene therapy applications but have not been widely tested. Promoters have also been identified that target gene expression to TM or Schlemm’s canal (SC). Studies of TM gene expression by sequencing of cDNA clones identified the matrix Gla protein, a member of a potassium-dependent protein that is widely expressed in bone, heart, kidney, and lung.24 VE-cadherin, which modulates cell–cell adhesion between vascular endothelial cells, was identified using spe-cific antibodies to be a marker for SC cells, but not TM cells.25

DNA sequence elements can be incorporated into the viral vector backbone as ‘enhancers’ of translation of pro-teins coded by the inserted transgene of interest. For example, hepatitis B viruses are known to regulate and enhance levels of protein synthesis in infected cells due to a post-translational regulatory element in the 3’ untrans-lated region of the hepatitis viral genome. A similar sequence has been isolated from woodchuck hepatitis virus, termed the woodchuck post-translational regulatory element (WPRE). WPRE increases viral RNA stability and is required for the cytoplasmic accumulation of viral RNAs.26 Incorpo-ration of the WPRE enhancer into rAAV vectors causes approximate 10-fold increases in the number of transfected neuronal cells and in protein expression as measured by Western blot, when compared to vectors without WPRE.27,28

The gene therapy approaches detailed above relies on transfection of a host cell by the viral vector, and expression of the transgene of interest relies on the host cell to modulate release of the gene product. The laminar protein fibronectin is secreted through a non-regulated constitutively active pathway, and inclusion of the fibronectin secretory signal sequence (FIB) in an AAV vector caused significant gene product secretion in vitro.29 A novel secretion strategy has been developed in which the FIB secretion signal sequence is fused with a transgene coding for a therapeutic peptide, causing marked enhancement of protein expression and positive therapeutic effect in a rat seizure model.30

Once viral DNA is transcribed by RNA polymerase into mRNA, protein translation by the ribosome begins at a unique site bound to 5’-end of the mRNA, termed the 5’ cap. Viral vectors have been designed to express

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have been shown to reduce choroidal neovascularization in a mouse model of age-related macular degeneration (AMD) and are currently in Phase 1 trials in humans.38

Modulation of Aqueous Outflow

Injection of viral vectors into the anterior chamber is an efficient technique to express transgenes specifically in the TM, because aqueous outflow causes deposition of vector primarily in this location. Gene delivery primarily using adenoviral or lentiviral vectors has demonstrated success in TM transfection and in modulation in aqueous outflow, usually by targeting extracellular matrix or cytoskeleton proteins. Recent success using siRNA therapy to target TM gene expression is an exciting development as well.

Anterior chambers of perfused porcine and human ante-rior segment cultures were injected with replication-deficient adenoviral constructs coding for reporter genes such as LacZ, luciferase, or GFP (Fig. 64-4).40,41 Transduc-tion of all layers of the TM (uveal, corneoscleral, and juxta-canalicular) and inner wall of SC was noted. Injections of high titers of vector were noted to cause an increase in outflow facility, possibly due to constipation of outflow channels with viral particles.40 TM cultures were trans-duced by adenovirus coding for aquaporin-1, an integral

six-member morpholine rings instead of five-member deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates.36 Advantages of morpholino antisense oligos are high affinity, nuclease resistance and ease of permeating cell membranes.37

Short interfering or silencing RNAs (siRNAs) are small (20–25 bp) double-stranded RNA molecules that are currently being explored for use in a gene therapy. RNA interference (RNAi) is an evolutionarily conserved pathway whereby siRNA silences gene expression post-transcriptionally. In the cytoplasm, siRNAs interact with a nuclease-containing RNA-induced silencing complex (RISC). Upon binding to RISC, the double-stranded siRNA unwinds, pairs with its complementary target mRNA, and allows the RISC complex to cleave the mRNA strand within the target site. This cleavage causes degradation of the mRNA molecule, preventing protein translation.38 Standard transfection methods can be employed to introduce exoge-nous siRNAs into cells to allow silencing of a specific gene of interest. As siRNA gene silencing is transient, the siRNA can be continuously expressed by an engineered plasmid vector. Drawbacks of the siRNA technique include immune responses due to siRNA overexpression and mistaken host recognition as a viral sequence. Treatments of ocular disease have shown promise, as adenovirus vectors coding for siRNA targeting vascular endothelial growth factor (VEGF)

Figure 64-4 Histochemical evaluation of β-galactosidase activity in the trabecular meshwork of perfused human anterior segment cultures. (A) Trabecular meshwork cells 48 h after transfection with Ad-lacZ. (B, C) Juxtacanalicular trabecular meshwork cells transfected with Ad-lacZ and stained with X-gal (large dark areas). (D) Trabecular meshwork cells treated with control vehicle do not stain with X-gal. (From Borras T, Rowlette LL, Erzurum SC, et al. Adenoviral reporter gene transfer to the human trabecular meshwork does not alter aqueous humor outflow. Relevance for potential gene therapy of glaucoma. Gene Ther, 1999; 6:515–24.)

A B

SC

C D

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nonhuman primates lays the groundwork for application of TM lentiviral vector transduction to human glaucoma gene therapy.52 Prostaglandin synthesis in TM was potenti-ated in the TM of cats after anterior chamber injection of a FIV-based lentiviral vector coding for the prostaglandin PGF2α receptor and cyclooxygenase-2, a rate-limiting enzyme in prostaglandin biosynthesis. Enhanced synthesis of prostaglandins using lentiviral gene therapy produced substantial and sustained reductions in IOP.53

Human perfused anterior segments were treated with short interfering RNA (siRNA) targeting matrix Gla protein (MGP). MGP transcripts as measured by reverse transcriptase-PCR (RT-PCR) were reduced by at least 93% in these eyes.38 In a separate experiment, glucocorticoid receptor (GR) siRNA was perfused for 48 h, followed by per-fusion with dexamethasone for 24 h. Dexamethasone is a potent steroid known to bind to GR and induce expression of myocilin, a gene associated with juvenile open-angle glaucoma.54 A reduction of MGP and myocilin gene expres-sion was noted by RT-PCR, and protein expression down-regulation was confirmed by Western blot.38 This experiment suggests that the IOP-elevating effects of steroids could be blocked by pre-treatment with GR siRNA. In another study using siRNA, mice were treated with dexamethasone to elevate IOP, and RhoA siRNA was injected into the anterior chamber. siRNAs accumulated in mouse TM in a dose-dependent manner, and caused large decreases in TM expression of RhoA mRNA and protein (p < 0.01). In dexamethasone-treated mice, IOP was significantly reduced from days 2 to 5 (p < 0.05).55

scAAVs are modified AAVs that bypass the required second-strand DNA synthesis to achieve transcription of the transgene. Self-complementary AAV (scAAV) generates a viral genome carrying both sense and complimentary cDNA of the transgene. On entry into the host, both strands pair with each other, generating the AAV double-stranded DNA needed for transcription.56,57 Single doses of AAV2 and scAAV coding for GFP were injected into the anterior chamber of rats and monkeys. While no GFP transduction was observed in TM of AAV-injected rats, scAAV2 caused efficient, long-lasting and well-tolerated TM transduction. This study represents the first successful transduction of TM using modified AAV vectors.58

Neuroprotection of Retinal Ganglion Cells

Due to the high efficiency of AAV vectors to transduce RGCs after intravitreal injection, the majority of studies attempt-ing neuroprotection have employed AAV to deliver trans-genes of interest to the retina. Initial studies using AAV to deliver reporter genes such as GFP or lacZ demonstrated no inflammation and persistence of transgene expression for up to 12 months.58,59 Neurotrophins and their receptors were among the first transgenes explored in neuroprotec-tive gene therapy experiments. TrkB is a receptor for the neurotrophin brain-derived neurotrophic factor (BDNF), which is important for RGC homeostasis. In an experimen-tal monkey glaucoma model, interruption of BDNF retro-grade transport and accumulation of TrkB at the optic nerve head was noted, suggesting a role for neurotrophin

membrane protein that functions as a channel for water and ion transport. Increases in mean resting cell volumes and paracellular permeability were seen in monolayers of TM cells, suggesting that aquaporin-1 is a modulator of outflow facility in vivo.42

Human TM cultures and rat anterior segments were noted to be efficiently transduced with replication-deficient adenovirus coding for stromelysin, a connective tissue degrading enzyme of the matrix metalloproteinase (MMP) family.43 Human TM and SC cell cultures as well as perfused human anterior segment cultures were transduced with an adenoviral construct coding for RhoA, a member of the Ras superfamily of GTP-binding proteins known to regulate cytoskeleton–cell interactions and endothelial barrier func-tions. Histological evidence of disruption of cytoskeleton components and endothelial cell adhesions was noted in cell culture, as well as a significant 32.5% mean increase of outflow facility in anterior segment culture after 72 h.44

Human TM cell cultures and perfused monkey eye ante-rior segments were transduced with adenovirus delivering exoenzyme C3 transferase, a member of the Rho GTPase family derived from Clostridium botulinum. Alterations in the cytoskeleton components actin, vinculin and β-catenin were noted in cell culture, as well as a mean 90% increase in outflow facility in monkey anterior segment culture.45 Human TM cell cultures and perfused monkey and human eye anterior segment cultures were transduced with adeno-virus delivering caldesmon, a regulator of myosin activity in smooth muscle cells. Changes in actin cytoskeleton and matrix adhesions were noted in TM cultures, and outflow facility was increased in anterior segment cultures of human eyes by a mean of 43% and in monkey eyes by a mean of 35% at 3 days and 66% at 6 days.46 A glucocorticoid-inducible adnenoviral vector overexpressing recombinant matrix metalloproteinase 1 (MMP1) was shown to reduce IOP in dexamethasone-treated perfused human anterior segment organ cultures, allowing for the development of gene therapy to steroid-induced ocular hypertension in human patients.47

Lentiviral gene therapy to increase outflow facility has great potential to lower IOP. Lentiviral vectors derived from feline immunodeficiency virus (FIV) have been shown to efficiently transduce TM cells for up to 10 months after anterior chamber injection, with minimal inflammation and cell loss.48,49 The same group of researchers also showed that a second-generation FIV vector caused an approximate 80% transduction of TM cells in perfused human anterior segment cultures, with a transient 30% decrease of outflow facility that resolved after 72 h.50

Dual-gene feline immunodeficiency virus (FIV) vectors using both 5′ cap-translation and IRES-translation were injected into the anterior chambers of domestic cats. Sub-stantial transgene expression of GFP and myocilin was seen in vivo for 1.2–2.3 years, demonstrating safe, long-term single and dual gene expression in TM after single injection of lentiviral vectors.51 FIV vectors encoding GFP were injected into the anterior chamber of cynomolgus monkeys, and GFP fluorescence in the TM was noted by gonioscopic monitoring for up to 15 months. Transduced cells were also detected in the iris and ciliary body. Mild but transient inflammatory responses were observed postinjec-tion, but IOP was not elevated. This initial study in

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Mean axon survival was significantly increased in the AAV-BDNF-WPRE group (67.7%) when compared to the control AAV-GFP-WPRE group (47.7%).62 These studies show the potential for neuroprotection of injured RGCs using neuro-trophin replacement by AAV gene therapy.

Modulation of programmed cell death, or ‘apoptosis’, is an attractive strategy for neuroprotection, as RGCs are known to die by apoptosis in human glaucoma.63 Endog-enous inhibitors of apoptosis proteins (IAPs) are evolution-arily conserved proteins known to directly inhibit caspases, the effector proteases of the apoptotic cascade.64 Rats were given unilateral intravitreal injections of AAV-CBA vector coding for human baculoviral IAP repeat-containing protein-4 (BIRC4), a potent caspase inhibitor. Ocular hyper-tension was induced in the same eye by sclerosis of aqueous humor outflow channels with hypertonic saline.65 After 12 weeks of chronic exposure to elevated IOP, optic nerve axon counts were performed to determine the neuroprotective effects of retinal BIRC4 expression, and axon survival was compared between AAV-BIRC4 and control AAV-GFP

deprivation in the pathogenesis of RGC death in glaucoma.60 After optic nerve axotomy (an acute injury model in which 90% of RGCs die by 2 weeks), TrkB mRNA was noted to decrease to approximately 50% of levels seen in intact retinas.61 AAV vector incorporating a CMV promoter was used to direct expression of either TrkB or GFP (control) in RGCs after intravitreal injection in rats. One week later, the optic nerves of the treated eyes were axotomized. Two weeks after axotomy, RGC survival was compared between the control AAV-GFP eyes and the AAV-TrkB eyes augmented with exogenous BDNF administration. Survival in the AAV-TrkB/BDNF group was 76%, compared to less than 10% in the AAV-GFP group.61 In a similar experiment, AAV incor-porating the CBA promoter and WPRE was used to direct expression of BDNF or GFP in RGCs after intravitreal injec-tion in rats (Fig. 64-5). Two weeks later, argon laser was applied to the trabecular meshwork, causing aqueous humor outflow obstruction and subsequent IOP elevation. After 4 weeks of elevated IOP exposure, RGC survival was assessed by axon counting of optic nerve cross-sections.

Figure 64-5 Transfection of rat retina with AAV-CBA-GFP-WPRE vector. (A) Individual RGCs and their axons clearly express GFP. (B) Dendritic trees of RGCs are also clearly seen. (C) GFP-labeled cells are localized almost exclusively to the RGC layer in retinal cross-sections. (D) In the immediate vicinity of the injection site, inner retinal cells are also transfected. RGC: retinal ganglion cell; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PR: photoreceptors. (From Martin KR, Klein RL, Quigley HA. Gene delivery to the eye using adeno-associated viral vectors. Methods, 2002; 28:267–75.)

A B

C D

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24. Gonzalez P, Caballero M, Liton PB, et al. Expression analysis of the matrix GLA protein and VE-cadherin gene promoters in the outflow pathway. Invest Ophthalmol Vis Sci 2004;45:1389–95.

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groups. AAV-BIRC4 gene therapy significantly promoted mean optic nerve axon survival (49.7%) when compared with AAV-GFP (22.3%).23 Following optic nerve axotomy in rats, RGC death is preceded by an early upregulation of the pro-apoptotic protein Bax.66 After injection of antisense Bax oligonucleotides in the temporal superior retina of rats, the number of RGCs surviving 8 days after optic nerve transection was increased when compared to eyes receiving control oligonucleotides. However, this neuroprotective effect was not seen at 14 days, suggesting that limitations in transfection efficiency and short half-lives limit the use of antisense oligos for neuroprotection of RGCs.67

Intracellular signaling pathways involved in cell death are also targets for neuroprotection of RGCs using AAV gene therapy. The transcription factor Max is found mostly within the nucleus of normal cells, and undergoes exclu-sion from the nucleus early in the apoptotic cascade in RGCs.68 AAV was used to overexpress Max in rat RGCs after intravitreal injection. Cell morphology was preserved in retinal explants from eyes treated with AAV-Max, when compared to retinal explants from uninjected control eyes.69 The extracellular signal-regulated kinase (Erk) 1/2 pathway is an evolutionarily conserved mechanism used by several peptide factors to promote cell survival. AAV coding for MEK1, an upstream activator of Erk1/2, was injected into rat eyes 3 weeks prior to IOP elevation induced by episcleral injection of hypertonic saline. After 7 weeks of exposure to chronic IOP elevation, AAV-MEK treatment significantly increased mean RGC survival (1366 RGCs/mm2 retina) when compared to AAV-GFP treatment (680 RGCs/mm2).70 Basic fibroblast growth factor (FGF-2) is a member of the fibroblast growth factor (FGF) family of neurotrophic factors that are involved in neuronal survival and synaptic plasticity. FGF-1 is known to be a potent stimulator of devel-opmental RGC axon growth and is correlated with activa-tion of the Erk1/2 pathway. Adult rat eyes were given intravitreal injections of AAV coding for FGF-2, followed by partial optic nerve crush injury. Although only transient neuroprotection of RGCs was noted, FGF-2 gene transfer led to a 10-fold increase in the number of axons extending 0.5 mm distal to the lesion site when compared to control nerves. These exciting findings show that factors mediating axon outgrowth during development may promote regen-eration of injured adult RGC axons.71,72

Erythropoietin (EPO) is a potential neuroprotective drug, but can produce polycythemia. By altering a single amino acid (R76E), EPO maintains its neuroprotective properties but does not alter erythropoiesis. A single intramuscular injection of AAV2 coding for this mutant form of EPO was given in DBA/2J mice, and preservation of RGCs, optic nerve axons and visual function was seen.73 For further insight, an excellent review of the current status of gene therapy for RGC neuroprotection in glaucoma is given by Wilson and Di Polo.74

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