adeno - associated viruses ( aav ) are a family of parvoviruses with a 4.7  kb single - stranded dna genome contained inside a non - enveloped capsid . the viral genome has 2 inverted terminal repeats ( itr)which function as the viral origin of replication and packaging signal  flanking 2 primary open reading frames ( orf ) : rep ( encoding proteins that function in viral replication , transcriptional regulation , site - specific integration , and virion assembly ) and cap . the cap orf codes for 3 structural proteins that assemble to form a 60-mer viral capsid . many naturally occurring aav variants and serotypes have been isolated , and none has been associated with human disease . additionally , recombinant versions of aav can be used as gene delivery vectors , where a marker or therapeutic gene of interest is inserted between the itrs in place of rep and cap . these vectors have been shown to transduce both dividing and non - dividing cells in vitro and in vivo and can result in stable transgene expression for years in post - mitotic tissue . differences in the protein capsid sequences of the natural aav serotypes are associated with different gene transfer properties . therefore , strategic modifications to the cap gene have the potential to yield delivery properties that are distinct from , and potentially highly advantageous compared to , any of the natural aav variants . while rational design has the potential to increase the efficiency of aav vectors , incomplete knowledge of viral delivery mechanisms coupled with the vast range of possible sequence modifications argues for unbiased , high - throughput algorithms for aav engineering . in particular , we have developed directed vector evolution as a high - throughput molecular engineering approach to successfully generate novel ,  designer aav variants with specific sequence modifications that enable them to overcome formidable barriers to efficient gene delivery in a broad range of applications . within this process , highly diverse libraries composed of 1010 genetic variants of the aav cap gene are iteratively subjected to selective pressures , resulting in enhanced gene delivery properties such as altered receptor binding , neutralizing antibody - evasion capabilities , and novel cell tropism . directed evolution has for example been applied to create novel aav vectors designed to overcome gene delivery barriers and/or preferentially transduce specific cell types in the central nervous system . for instance , because natural aav serotypes preferentially transduce neurons , research has also been undertaken to develop aav vectors that are capable of efficient transduction of other neural cells . utilized a diverse library of aav variants generated through dna shuffling , random mutatgenesis , and surface loop replacement to evolve vectors for the ability to infect primary human astrocytes in culture . the resulting vectors transduced between 2-fold and 15-fold more primary human astrocytes compared to aav serotype 2 ( aav2 ) . two variants tested in vivo also transduced 3.3-fold and 5.5-fold more astrocytes than wild - type aav2 within the rat striatum following intracranial injection . these 2 variants display similar peptide motifs at amino acid 262 ( in loop 2 of the aav capsid ) , which may enhance cell binding or internalization . using a library of shuffled cap genes from several wild - type serotypes , gray et  al . isolated aav variants capable of gaining access via intravenous administration to regions of the brain in which seizure had compromised the blood  brain barrier . after kainic acid - induced seizure , 2 clones composed primarily of wild - type aav1 , 8 , and 9 could transduce cells in the piriform cortex and ventral hippocampus of rats upon tail vein administration of the vectors , but no transduction occurred in brain areas where the blood  brain barrier was not compromised . within these brain areas , comparison of the in vivo biodistribution of these clones with parental serotypes aav1 , 8 , and 9 revealed that the portion of aav8 within these clones likely led to the ability to cross the seizure compromised barrier , while the aav1 portions of the clones likely led to peripheral organ transduction pattern . in addition to efforts to target largely postmitotic cells , jang et  al . applied directed evolution to engineer an aav variant capable of efficient neural stem cell ( nsc ) transduction . using a cap library generated by dna shuffling , random mutagenesis , and random peptide insertion , selection for the ability to infect cultured adult hippocampal nscs yielded aav r3.45 , an aav2 variant with a 7 amino acid peptide insertion at position 588 . compared to wild - type aav2 , aav r3.45 mediated 50-fold increased transduction of rat nscs and significantly increased transduction of murine nscs , human fetal nscs , and human embryonic stem cell ( hesc ) derived neural progenitor cells in vitro . a subsequent study showed that within an hesc - derived culture containing a mixture of cells including nscs , neurons , and astrocytes , the percentage of nscs infected by aav r3.45 was significantly higher compared to wild - type aav . the variants still bound heparan sulfate proteoglycans ( hspg , aav2s primary receptor ) , though peptide insertion at amino acid 588 in the parental aav2 capsid lowered aav 3.45s hspg affinity . in addition , the inserted peptide , which did not have homology to any known cell surface proteins , likely conferred affinity to an undetermined secondary receptor that potentially mediated viral internalization . aav r3.45 also efficiently and preferentially transduced rat and mouse nscs following intracranial administration to the hippocampus . approximately 65% of the cells transduced in the rat hippocampus by aav r3.45 were type 2a nscs , and 9% were type 1 nscs . furthermore , 60% of type 2a nscs and 41% of the type 1 nscs , respectively , were transduced by aav r3.45 in the rat brain . similarly , in the mouse brain approximately 38% of type 2a nscs were transduced in the hippocampus . there has also been considerable work in engineering novel aavs for delivery to the neuroretina . the cells most commonly involved in retinal disease , photoreceptors and retinal pigment epithelium , are located in the outer retina , separated from the vitreous fluid of the eye by the inner limiting membrane and several dense cell layers . as a result , no natural aavs are capable of transducing these cells following an intravitreal injection , a preferred , noninvasive route of administration that could conceivably enable vector to transduce the entire retinal surface area . therefore , clinical trials conducted to date  which have been increasingly successful for leber 's congenital amaurosis type 2 and choroideremia  have relied on vector administration via subretinal injection , a surgery that can damage both healthy and diseased retinas and that results in transduction in a relatively small portion of the retina . two vectors capable of improved outer retinal transduction upon intravitreal injection have recently been developed in rodent . engineered an aav variant , shh10 , capable of highly specific ( over 94% of transduced cells ) and efficient infection of mller cells , glial cells that span the thickness of the retina , when delivered intravitreally . further analysis of the individual point mutations in shh10 revealed that its n451d mutation decreased the variant 's dependence on n - linked sialic acids , and this single point mutation was sufficient to cause intravitreal mller cell tropism . in addition , the d532n mutation appears to increase affinity to hspg , which are not a receptor for the parental aav6 . in a subsequent study , this vector mediated broad expression of glial cell - derived neurotropic factor ( gdnf ) , which slowed retinal degeneration in a rat model of retinitis pigmentosa . in another study , using in vivo directed evolution in a mouse , dalkara et  al . generated the aav variant 7m8 , which was capable of transporting genetic cargo through the retina and directly infecting photoreceptors after intravitreal delivery . the peptide insertion at amino acid 588 in the parental aav2 capsid lowered 7m8s affinity for hspg , though infectivity was still hspg - dependent . in addition , the 7 amino acid peptide insertion at position 588 mediated substantially higher gene expression in mouse photoreceptors in vivo compared to wild - type aav , and led to the functional rescue of murine models of x - linked retinoschisis and leber 's congenital amaurosis type 2 . ranging from optogenetics to site - specific nucleases  to investigate basic neuroscience problems including neurodevelopment , adult neurogenesis , brain activity mapping , learning and memory , cns aging , and human neurological disease biology . as model systems for investigation in each of these areas , transgenic and mutant mice offer the capacity to study the functional effects of targeted gene modifications in an in vivo environment . for example , in the area of adult neurogenesis , transgenic mouse lines such as the nestin - creer mouse have enabled a number of basic advances . however , deriving new lines to study each new gene is highly time and labor intensive , taking months to years to create , and their application to study phenotypes associated with aging or neurodegenerative disease models adds considerably more time . is not available in other organisms , such as large animals . as a versatile and powerful alternative , efficient and selective gene delivery to specific cell types within compared to a months - to - years timeframe to generate ( and , if relevant , age ) a new mouse , studies involving targeted gene delivery with a wild type mouse can proceed from design to data in a matter of weeks , even taking into account the time needed for surgery . for example , different studies have implicated wnt/-catenin signaling in both the self - renewal and the lineage commitment of adult hippocampal stem cells , ostensibly contradictory results . to address this question , the nsc - selective aav r3.45 was harnessed to deliver constitutively - active -catenin ( ca -catenin ) in the mouse hippocampus . the result was significant increases in the number of nscs , neural progenitor cells , and neurons in the hippocampus , indicating that -catenin signaling increases neurogenesis via modulating both proliferation and differentiation of nscs . this first use of an engineered , targeted aav to investigate neurogenesis presages future studies with , for example , astrocyte or nsc - selective vectors to study cell - extrinsic and cell - intrinsic mechanisms that control nsc quiescence , proliferation , self - renewal , and differentiation . in the retina , the mller cell - tropic aav vector shh10 was utilized to study the role of glial cell secretion of ciliary neurotrophic factor ( cntf ) in axonal regeneration . shh10-mediated expression of cntf was sufficient to promote long - distance regeneration of severed optic axons in a mouse optic nerve crush trauma model . the efficient and selective transduction of glial cells in the retina and subsequent cntfr secretion resulted in sustained regeneration and protection of axons in the optic nerve . this study also reported previously unexplored aspects of the mechanism of cntfr - mediated neuronal growth , including the induction of massive ectopic sprouting of axons in the retina and axonal misguidance in the absence of additional guidance signaling . aav variants shh10 and 7m8 were also used to elucidate the mechanism by which cellular retinaldehyde - binding protein ( cralbp ) supports cone photoreceptor function . in particular , xue et  al . utilized shh10-mediated expression of cralbp in mller cells and 7m8-mediated expression of cralbp in retinal pigment epithelia in mice lacking the rlbp1 gene ( which encodes cralbp ) to determine that rescuing cralbp expression specifically in mller cells but not in retinal pigment epithelia improved m - cone sensitivity and restored the retinal visual cycle in these mice . different natural and engineered aav variants have delivery properties that vary by cell type , tissue target , and species ; therefore , the variant must be carefully chosen to suit a given application . for example , although different natural aav serotypes share some highly conserved capsid regions , there are 9 hypervariable regions that result in differences in cell surface receptor binding and tissue tropism . in a study comparing the biodistribution of aav19 following tail vein administration to mice , differences in both transduction efficiency and specificity of the various serotypes could be observed . for example , aav2 transduction resulted in 26- to 30-fold more gene expression from the liver compared to the skeletal muscle and heart , respectively . aav6 gene expression from the heart was 2.7- to 4.7-fold higher than gene expression in the skeletal muscle or liver . aav9 broadly transduced the heart , liver , lungs , and skeletal muscle tissues evaluated . furthermore , the transduction efficiencies and biodistributions of various aav serotypes differ between animal species . in mice and non - human primates , however , in clinical trials for hemophilia b , aav2 and aav8 vectors mediated similar peak levels of factor ix protein expression in patients . these species differences are also seen with engineered aav vectors . despite strong transduction in the mouse retina using the variant 7m8 , infection in the non - human primate retina was limited . these proof of concept studies in mouse thus illustrate another consideration for directed evolution : gene delivery barriers change with species , and thus vectors engineered for optimized delivery in rodents may not translate to large animals or to patients . however , future engineering in large animal models may yield vectors with a strong translational path to human clinical studies . in addition to engineering aav variants for specificity to certain cell types or tissues as described above , another mechanism for restricting gene expression to a cell type of interest is to engineer the payload being delivered . endogenous , cell - type specific promoters have commonly been used to restrict expression to specific cells . for example , a rhodopsin promoter can mediate aav - mediated gene expression specifically in rod photoreceptors . alternatively , the incorporation of short target sequences for tissue - specific microrna into the transgene cassette can be used to detarget gene expression from specific tissues . when cells that express the corresponding microrna are transduced , binding of the microrna to the vector payload functions to downregulate expression of the transgene . this strategy has been employed to show that expression from aav vectors can be reduced using liver - specific , heart - specific , and antigen presenting cell - specific microrna . the use of promoters or microrna regulatory elements to express the transgene only from the cell type of interest will help reduce off - target gene expression or possible immune responses to the transgene resulting from antigen presentation of the therapeutic protein . the use of endogenous promoters can also serve to decrease the level of expression in cells of interest compared to stronger viral promoters , which could in some cases maintain transgene expression within a therapeutic efficacy window . in addition to basic studies , gene delivery can be harnessed for gene or cell replacement therapies to treat neurodegenerative disease or injury . a number of cns clinical trials have established safety but not strong evidence for efficacy to date , and enhanced vectors may aid future clinical efforts . for example , delivery of aspartoacylase ( for canavan 's disease ) and cln2 ( for late infantile neuronal ceroid lipofuscinosis ) transgenes using natural aav have been established as safe . use of an engineered vector that achieves more efficient spread and thus broader transduction of the central nervous system could improve efficacy for these monogenic disorders . aav - mediated delivery of transgenes such as aromatic l - amino acid decarboxylase ( aadc ) , glutamic acid decarboxylase ( gad ) , and glial derived neurotrophic factor ( gdnf ) have been explored in clinical trials for parkinson 's disease , though these trials have not yet achieved the efficacy necessary to become a treatment , potentially due to limited delivery efficiency . in one study , the aav2 vectors used in the aadc clinical trial transduced only 56% of neurons near the injection site in the nonhuman primate brain . use of an engineered vector for higher infectivity and broader distribution could again yield higher efficacy in such studies . analogously , within the eye , subretinal injections of aav2 vectors have yielded highly promising results in clinical trials for leber 's congenital amaurosis type 2 and choroideremia . however , many additional retinal diseases that are candidates for gene therapy feature heavily damaged retinas that would benefit from non - invasive delivery via the vitreous , which can also offer broader expression across the retina compared to a subretinal injection . engineered aav variants are needed for outer retinal transduction from the vitreous . as a final example of future potential applications , inducible , conditional expression of the fmr1 gene in nscs resulted in restoration of fragile x mental retardation protein expression specifically in adult nscs and rescued mice from learning deficits in a murine model of fragile x syndrome . use of an nsc - specific gene delivery vector could offer a path for clinical translation . gene delivery vectors based on adeno - associated virus ( aav ) have emerged as effective research tools and promising clinical vectors . although the central nervous system presents many barriers to delivery , novel , engineered aav vectors capable of more efficient delivery , more specific targeting , and broader transduction are increasingly helping to surmount these challenges and further enable biomedical research in the cns .
adeno - associated viruses ( aav ) are non - pathogenic members of the parvoviridae family that are being harnessed as delivery vehicles for both basic research and increasingly successful clinical gene therapy . to address a number of delivery shortcomings with natural aav variants , we have developed and implemented directed evolution  a high - throughput molecular engineering approach to generate novel biomolecules with enhanced function  to create novel aav vectors that are designed to preferentially transduce specific cell types in the central nervous system ( cns ) , including astrocytes , neural stem cells , and cells within the retina . 
 these novel aav vectors  which have enhanced infectivity in vitro and enhanced infectivity and selectivity in vivo  can enable more efficient studies to further our understanding of neurogenesis , development , aging , and disease . 
 furthermore , such engineered vectors may aid gene or cell replacement therapies to treat neurodegenerative disease or injury .