Virus used for gene therapy

Request an Appointment at Mayo Clinic. Share on: Facebook Twitter. Show references Gene therapy. Genetics Home Reference. Accessed July 21, What is gene therapy? National Cancer Institute. Accessed July 22, Chuah M, et al. Recent progress in gene therapy for hemophilia. Human Gene Therapy. Cicalese M, et al. Clinical applications of gene therapy for primary immunodeficiencies. Alphaviruses possess an ssRNA genome encapsulated in a capsid and envelope protein structure [ 23 ].

The nonstructural proteins nsPs expressed from alphavirus vectors form the replicon complex generating extreme RNA replication. The self-amplifying nature of the positive sense genome makes alphavirus vectors attractive for short-term transient heterologous gene expression [ 24 ]. In the case of RNA and DNA-based expression, the gene of interest is introduced into the alphavirus expression vector downstream of the nsP genes, whereas viral particle production requires co-delivery of the alphavirus structural genes located on a helper vector [ 25 ].

The insert capacity of alphavirus vectors is 8 kb, but also multiple delivery with different constructs is feasible. Moreover, the naturally occurring oncolytic M1 virus has been applied for cancer therapy [ 28 ].

One drawback of application of recombinant alphavirus particles relates to the lack of an efficient packaging system. Although packaging cell lines have been engineered for SFV and SIN [ 29 ], and XJ virus [ 30 ], the titers are not compatible with high-titer virus production at large scale. Related to the KUN expression vector, the gene of interest is inserted between the C20 core protein and the E22 envelope protein in frame with the viral polyprotein.

Moreover, to facilitate virus production a packaging cell line has been engineered for KUN [ 32 ]. In addition to KUN, expression systems have been developed for West Nile virus [ 33 , 34 ], yellow fever virus [ 35 , 36 ], Dengue virus [ 37 , 38 ], and tick-borne encephalitis virus [ 39 , 40 ].

In contrast to alphaviruses and flaviviruses, rhabdoviruses carry a negative strand ssRNA genome [ 41 ]. Due to the negative polarity of RNA, efficient transgene expression has been achieved by application of reverse genetics based on a recombinant vaccinia virus.

Similar to rhabdoviruses, measles viruses MV possess an ssRNA genome of negative polarity, which has required the design of rescue systems by reverse genetics for replicating MV from cloned DNA in a helper cell line [ 47 , 48 ]. MV expression vectors have been engineered with the gene of interest inserted either between the phosphoprotein P and the matrix protein M or between the hemagglutinin HA and the large protein L.

Recombinant MV particles can be generated by transfection of a helper cell line with recombinant MV constructs and a plasmid carrying the MV polymerase L gene. NDV offers several advantages due to its host range restriction and no pre-existing immunity in the human population.

High titer NDV replication can be achieved in cell lines acceptable for human vaccine development. In an attempt to optimize foreign gene expression from NDV vectors, reverse genetics was applied to introduce the red fluorescence protein RFP at various sites downstream of the nucleocapsid NP , P, M, fusion F , hemagglutinin-neuraminidase HN , and large polymerase L genes [ 51 ].

This allows regulation of transgene expression by selecting the insertion site of the gene of interest. One feature that has made NDV attractive for cancer therapy relates to the specific replication in tumor cells [ 53 ]. The CVB3-renilla was completely attenuated and demonstrated superior expression levels in mice in comparison to adenovirus-based expression [ 54 ]. Moreover, coxsackieviruses have been applied as oncolytic vectors in cancer therapy [ 56 ].

Due to the large number of preclinical studies on gene therapy and vaccines conducted with RNA viruses in various animal models only some examples are presented below. Moreover, gene therapy and vaccine applications in animal studies are listed in Table 2. The classic approach for long-term gene expression has been to apply retrovirus vectors such as amphotropic retroviral vectors containing the mutant dihydrofolate reductase gene DHFR or the bacterial neomycin phosphotransferase neo gene for transduction of canine hemopoietic cells as a model for human gene therapy [ 57 ].

Moreover, several retroviral vectors have shown efficient transduction of undifferentiated murine embryonic and hematopoietic cells, which promoted preclinical studies in murine models [ 58 ]. In the context of cancer therapy, the yeast cytosine deaminase CD was expressed from the nonlytic amphotropic retroviral replicating vector RRV Toca and evaluated in an orthotopic glioma mouse models [ 59 ]. Combination of RRV-Toca and 5-fluorocytosine 5-FC resulted in cell death and long-term survival in immune-competent mice.

Moreover, the self-inactivating gammaretroviral vector SINfes. Related to adoptive cell therapy for the treatment of cancer patients, retrovirus vectors have been used for gene modifications of natural killer cells [ 61 ]. Robust expansion of gene-modified NK cells was established, which will support production of GMP grade material for clinical trials.

Intradermal injection of genetically corrected RDEB fibroblasts reversed the disease phenotype in a xenograft model in nude mice. Related to Xeroderma pigmentosium, a skin disease caused by deficiency in nucleotide excision repair NER , a retrovirus-based strategy to provide XP-C keratinocytes with the wild-type XPC gene was evaluated in isolated keratinocyte stem cells [ 63 ].

No adverse effects such as oncogenic activation or clonal expansion were observed, and normal epidermal differentiation in both organotypic skin cultures and in a mouse model of human skin regeneration occurred. Due to their broad host range lentiviruses have been applied for many different disease areas.

Moreover, lentivirus-based siRNA delivery decreased tau phosphorylation and the number of neurofibrillary tangles in an AD mouse model [ 68 ]. Transplantation of lentivirus-transduced HSCs in myeoblated PKD mice normalized the erythroid compartment resulting in a corrected hematological phenotype and reversion of organ pathology.

Analysis of the chromosomal insertion site in transplanted HSCs showed no presence of genotoxicity. Moreover, secondary transplantations confirmed long-term safety showing no hematological or biochemical toxicity.

These findings provided a good basis for conducting clinical trials. In a highly promising approach, lentivirus vectors have been applied for chimeric antigen receptor T cell CAR-T technology in preclinical models of hematologic tumors [ 73 ]. All treatments provided rapid elimination of leukemia in a mouse model of acute myeloid leukemia AML. Moreover, CAR-T cell persistence prior to ablation was required for four weeks to achieve a durable leukemia remission.

EOC is a particularly interesting model for CAR-T due to the pattern of diffusion within the peritoneal cavity, the tumor microenvironment, and the high rate of tumor associated antigens. CAR-containing cells showed no toxicity and persisted for more than two years and generated multilineage engraftment and immune surveillance.

In another study, the Cal-1 anti-HIV lentiviral vector was evaluated for safety and efficacy in pigtailed macaques [ 77 ]. Robust levels of gene marking without measurable adverse events was observed in myeloid and lymphoid lineages confirming the safety of the process. Challenges with SHIV led to positive selection for gene-marked cells.

Self-amplifying alphavirus vectors have been subjected to numerous studies especially related to cancer gene therapy and vaccine development [ 78 ].

Local administration of replication-proficient SFV particles expressing enhanced green fluorescent protein EGFP prolonged survival of mice with implanted A lung carcinoma xenografts [ 79 ]. The naturally occurring M1 alphavirus has been evaluated in a liver tumor model, which demonstrated selective killing of zinc-finger antiviral protein ZAP -deficient cancer cells and potent oncolytic activity [ 82 ].

Self-amplifying alphaviruses have been further applied for vaccine development against HIV [ 86 ]. The humoral immune responses were superior for the immunization with SFV particles. Recombinant particles elicited stronger immune responses than RNA replicons. Inoculation in ovo and at one day of age resulted in partial protection, while complete protection was achieved after a single injection at two weeks of age.

In another study, SFV particles carrying the influenza HA and NP genes provided protection against lethal challenges with influenza virus in immunized mice [ 90 ]. Although mice were protected by both vaccination strategies, 64 times less self-amplifying RNA 1. Related to Lassa virus, a bi-cistronic VEE vector with two 26S subgenomic promoters expressing Lassa virus glycoproteins of distantly clades I and IV provided protection in mice against challenges with Lassa virus [ 92 ].

In the context of flaviviruses, some preclinical studies have been conducted in the area of cancer therapy. For instance, non-cytopathogenic KUN particles expressing the granulocyte colony-stimulating factor G-CSF were intratumorally injected into mice implanted with aggressive subcutaneous CT26 colon carcinomas and BOVA melanomas [ 93 ].

The treatment also generated regression of CT26 lung metastases. An interesting finding was recently reported that indicated that Zika virus possesses oncolytic activity in glioblastoma stem cells GSCs [ 94 ].

The preferential killing of GSCs seems to be specific for Zika virus as other flaviviruses such as West Nile virus indiscriminately killed both tumor and normal neuronal cells. It was also demonstrated that Zika virus potently depleted patient-derived GSCs in culture and mice with established glioblastomas survived significantly longer in a mouse-adapted Zika virus strain.

The glioblastoma targeting property of Zika virus may provide an attractive alternative for future therapeutic interventions. The constructs included the wild-type gag gene, an RNA-optimized gag gene, a codon-optimized gag gene, and a modified gag-pol gene construct. The KUN-SIV gag-pol vaccine demonstrated the best effector memory and central memory responses, and mediation of protection.

The concern of applying oncolytic VSV vectors for brain cancer relates to their inherent neurotoxicity. However, engineered pseudotyped VSV particles VSV-GP , where the VSV-G envelope has been replaced by the non-neurotropic envelope glycoprotein from the lymphocytic choriomeningitis virus LCMV , enhanced brain cancer cell susceptibility in vitro and were unable to infect primary human and rat neurons in vitro and in vivo, respectively [ 98 ].

Furthermore, the LCMV-pseudotyped VSV vector efficiently infected and killed all human, mouse, and canine melanoma cell lines tested as well as most human primary cultures [ 99 ]. Moreover, the survival was prolonged in both xenograft and syngeneic mouse models. Related to ovarian cancer, the pseudotyped VSV-GP vector generated oncolytic activity in ovarian cancer cell lines and in vivo [ ]. The response in both subcutaneous and orthotopic xenograft models could be enhanced by combination therapy with ruxilitinib.

The pseudotyped VSV-GP also provided long-term remission in prostate cancer mouse models after intratumoral injections [ ]. Furthermore, intravenous administration of subcutaneous tumors and bone metastases resulted in remission. The therapeutic applications of measles virus MV include immunization of MV vectors expressing the domain III of Dengue virus envelope protein 2 DV2 , which elicited robust neutralizing antibody responses in MV-susceptible mice [ ].

Furthermore, MV vectors displaying the domain III of DV generated both neutralizing antibody responses and provided protection against challenges with four Dengue virus serotypes in mice [ ]. In the context of vaccine development, complete protection against challenges with virulent NDV was obtained in specific-pathogen-free chicken immunized with the thermostable avirulent NDV strain TSC containing the GFP gene [ 52 ]. Related to NDV-based preclinical studies, the NDV strain T caused long-lasting complete tumor regression in athymic mice implanted with human neuroblastoma and fibrosarcoma xenografts [ ].

Although a single intratumoral or intraperitoneal injection generated complete regression of IMR neuroblastoma xenografts in 9 out of 12 mice, multiple administration provided superior efficacy. Moreover, a modified NDV vector containing the highly fusogenic F protein exhibited significant reduction in tumor development and prolonged survival in mice with implanted CT26 tumors [ ]. Intratumoral administration of NDV-F-IL-2 generated a dramatic decrease in tumor growth and the majority of treated animals showed complete and long-lasting remission.

Moreover, the anti-proliferative effect of the NDV D90 strain described in the human lung cancer cell line A was also evaluated in athymic mice bearing implanted lung tumors [ ].

The hybrid vector showed reduced neurotoxicity and was avirulent in embryonated chicken eggs. Moreover, systemic administration of rVSV-NDV showed significant prolongation of survival in immune-competent mice implanted with orthotopic hepatocellular carcinoma HCC. Coxsackievirus vectors have been frequently applied for gene therapy [ 56 ].

In this context, the Coxsackievirus B3 CVB3 expressing the human fibroblast growth factor 2 FGF2 showed protection from ischemic necrosis after administration into ischemic hindlimbs of mice [ ].

Despite being a relatively young field and the set-backs that were experienced, an impressively large number of clinical trials have been conducted and an increasing number of trials are planned or in progress.

A summary of trials is presented below and in Table 3. The classic example of retrovirus-based gene therapy trials relates to the treatment of children with SCID, which provided complete cure although development of leukemia occurred in some patients [ 3 , 4 ].

Since then, thorough vector development related to safety and efficacy issues has revitalized the application of retroviruses for clinical trials. For instance, Toca retrovirus vectors have been subjected to a clinical phase I trial in patients with recurrent high-grade glioma HGG [ ]. The overall survival rate of The results indicated that despite transiently resolved bacterial and fungal infections, clonal dominance and malignant transformations compromised therapeutic efficacy. Lentivirus-based clinical trials have been less common than those for retroviruses due to their later vector development.

These findings support the initiation of the first-in-man trial in CF patients. Lentiviruses have found applications in treatment of leukemia. A low dose of autologous chimeric antigen receptor-modified T cells 1. Furthermore, remission continued for 10 months. Moreover, the lentivirus-based chimeric antigen receptor-modified T CAR-T cells targeting CD19 therapy was evaluated in 30 children and adults with relapsed acute lymphoblastic leukemia ALL [ ].

Moreover, lentivirus-based transduction of stem cells differentiated to adipogenic, chondrogenic, and osteoblastic cells provided high level expression of factor IX applicable for hemophilia B treatment [ ]. These approaches resulted in enhanced dopamine and L-Dopa production. Engineering of a self-inactivating lentivirus vector expressing the sh5 anti-HIV gene and the C46 anti-viral fusion inhibitor peptide contributed to a synergistic effect on HIV-1 inhibition [ ].

Although the treatment was well tolerated, only modest local immune responses with low levels of binding antibodies and T cell responses was achieved. Repeated immunization elicited clinically relevant CEA-specific T cell and antibody responses and prolonged survival was obtained in patients with CEA-specific T cell responses. CRPC patients administered with either 0. Despite that neither robust immune responses nor clinical benefits were obtained, the presence of neutralizing antibodies indicated that dose optimization might improve the immunogenicity.

In attempts to provide passive tumor targeting, SFV particles were encapsulated in liposomes [ 24 ]. Moreover, the encapsulation substantially enhanced tumor targeting and prevented recognition by the host immune system after repeated administration. Clinical trials for rhabdoviruses have mainly been comprised of immunization studies for VSV vectors targeting infectious diseases, particularly EBOV. Although some adverse events such as injection site pain, fatigue, myalgia, and headache occurred the overall safety profile was good.

The lowest dose elicited lower antibody titers at day 28 compared to the two higher doses. Significantly increased antibody titers were observed after a second immunization on day 28, but the effect disappeared after six months.

Adverse events such as injection site reactions, headache, fever, and fatigue were more common in individuals receiving active vaccine in comparison to placebo. Among the participants individuals were assigned for immediate vaccination with VSV-EBOV and persons for delayed vaccination. No cases of EBOV were detected in the group receiving the vaccine at the start of the study after ten days, whereas 16 EBOV cases were discovered in the individuals receiving delayed vaccination.

Measles viruses have been subjected to clinical trials mainly in cancer therapy. Moreover, a Phase I trial in patients with advanced ovarian cancer was performed by intraperitoneal injection of 10 3 —10 9 MV-CEA showing no dose-limiting toxicity [ ].

Stable disease was observed in 14 patients with a median duration of 88 days and a range of 55— days. Related to the applied dose, 10 7 —10 9 TCID 50 resulted in stable disease in all patients, whereas it was accomplished for only five out of 12 with doses between 10 3 and 10 6 TCID Patients with relapsed refractory myeloma have been subjected to a Phase I clinical trial of intravenous administration of oncolytic MV vectors expressing the human sodium iodide symporter NIS [ ].

The patient remained disease-free for an additional 19 months due to an irradiation procedure. Newcastle disease virus have been subjected to several clinical trials in the area of cancer. For instance, expression of multiple tumor-associated antigens TAAs from NDV vectors has provided long-term survival in Phase II trials in ovarian, stomach, and pancreatic cancers [ ]. In another study, the NDV PV strain was intravenously administered to 79 patients with advanced solid tumors in a Phase II trial at a low dose of 1.

Administration of the higher dose resulted in objective responses and progression-free survival ranging from four to 31 months. In a more positive outcome, patients with colorectal cancer were immunized with NDV vectors in a Phase III trial, which prolonged survival and improved short-term quality of life in patients [ ]. Coxsackievirus vectors have been applied for clinical trials mainly for the treatment of melanoma.

Moreover, coxsackievirus vectors have been subjected to combination therapy. In this context, the antitumor activity of CVA21 was enhanced by co-treatment of melanoma patients with immune checkpoint blockade in a Phase II trial, which led to induced immune cell filtration in the tumor environment [ ]. Remember Me. Log in. Advertise on this site? Advertising opportunities on Gene Therapy Net include standard size banners as well as text ads. Click here for more information about advertising.

Viral Vectors All viruses attack their hosts and introduce their genetic material into the host cell as part of their replication cycle. This genetic material contains basic 'instructions' of how to produce more copies of these viruses, hijacking the body's normal production machinery to serve the needs of the virus see figure 1. The host cell will carry out these instructions and produce additional copies of the virus, leading to more and more cells becoming infected.

Some types of viruses actually physically insert their genes into the host's genome. This incorporates the genes of that virus among the genes of the host cell for the life span of that cell.

Risk Factors The concept of gene therapy seems straightforward, but this is clearly an oversimplification, and numerous problems and risks exist that prevent gene therapy using viral vectors. Viruses can usually infect more than one type of cell.

Thus, when viral vectors are used to carry genes into the body, they might infect healthy cells as well as cancer cells. Learn More. There has been a resurgence in gene therapy efforts that is partly fueled by the identification and understanding of new gene delivery vectors. Adeno-associated virus AAV is a non-enveloped virus that can be engineered to deliver DNA to target cells, and has attracted a significant amount of attention in the field, especially in clinical-stage experimental therapeutic strategies.

The ability to generate recombinant AAV particles lacking any viral genes and containing DNA sequences of interest for various therapeutic applications has thus far proven to be one of the safest strategies for gene therapies. This review will provide an overview of some important factors to consider in the use of AAV as a vector for gene therapy. The discovery of DNA as the biomolecule of genetic inheritance and disease opened up the prospect of therapies in which mutant, damaged genes could be altered for the improvement of the human condition.

The recent ability to rapidly and affordably perform human genetics on hundreds of thousands of people, and to sequence complete genomes, has resulted in an explosion of nucleic acid sequence information and has allowed us to identify the gene, or genes, that might be driving a particular disease state. This concept seems particularly true for the treatment of monogenic diseases, i.

This seemingly simple premise has been the goal of gene therapy for over 40 years. Until relatively recently, that simple goal was very elusive as technologies to safely deliver nucleic acid cargo inside cells have lagged behind those used to identify disease-associated genes.

One of the earliest approaches investigated was the use of viruses, naturally occurring biological agents that have evolved to do one thing, i. There are numerous viral agents that could be selected for this purpose, each with some unique attributes that would make them more or less suitable for the task, depending on the desired profile [ 1 ].

However, the undesired properties of some viral vectors, including their immunogenic profiles or their propensity to cause cancer have resulted in serious clinical adverse events and, until recently, limited their current use in the clinic to certain applications, for example, vaccines and oncolytic strategies [ 2 ]. More artificial delivery technologies, such as nanoparticles, i.

Not surprisingly, they also have encountered some unwanted safety signals that need to be better understood and controlled [ 3 ]. Adeno-associated virus AAV is one of the most actively investigated gene therapy vehicles. It was initially discovered as a contaminant of adenovirus preparations [ 4 , 5 ], hence its name.

AAV belongs to the parvovirus family and is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. Initially distinguished serologically, molecular cloning of AAV genes has identified hundreds of unique AAV strains in numerous species. These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing.

These coding sequences are flanked by inverted terminal repeats ITRs that are required for genome replication and packaging. It is estimated that the viral coat is comprised of 60 proteins arranged into an icosahedral structure with the capsid proteins in a molar ratio of VP1:VP2:VP3 [ 6 ]. The aap gene encodes the assembly-activating protein AAP in an alternate reading frame overlapping the cap gene. This nuclear protein is thought to provide a scaffolding function for capsid assembly [ 7 ].

Although there is much more to the biology of wild-type AAV, much of which is not fully understood, this is not the form that is used to generate gene therapeutics. In the absence of Rep proteins, ITR-flanked transgenes encoded within rAAV can form circular concatemers that persist as episomes in the nucleus of transduced cells [ 9 ]. Because recombinant episomal DNA does not integrate into host genomes, it will eventually be diluted over time as the cell undergoes repeated rounds of replication.

This will eventually result in the loss of the transgene and transgene expression, with the rate of transgene loss dependent on the turnover rate of the transduced cell. These characteristics make rAAV ideal for certain gene therapy applications. Following is an overview of the practical considerations for the use of rAAV as a gene therapy agent, based on our current understanding of viral biology and the state of the platform.

The final section provides an overview for how rAAV has been incorporated into clinical-stage gene therapy candidates, as well as the lessons learned from those studies that can be applied to future therapeutic opportunities.

The main point of consideration in the rational design of an rAAV vector is the packaging size of the expression cassette that will be placed between the two ITRs. As a starting point, it is generally accepted that anything under 5 kb including the viral ITRs is sufficient [ 10 ]. Attempts at generating rAAV vectors exceeding packaging cassettes in excess of 5 kb results in a considerable reduction in viral production yields or transgene recombination truncations [ 11 ].

As a result, large coding sequences, such as full-length dystrophin, will not be effectively packaged in AAV vectors. Therefore, the use of dual, overlapping vector strategies reviewed by Chamberlain et al. An additional consideration relates to the biology of the single-stranded AAV-delivered transgenes. After delivery to the nucleus, the single-stranded transgene needs to be converted into a double-stranded transgene, which is considered a limiting step in the onset of transgene expression [ 13 ].

An alternative is to use self-complementary AAV, in which the single-stranded packaged genome complements itself to form a double-stranded genome in the nucleus, thereby bypassing that process [ 13 , 14 ]. Although the onset of expression is more rapid, the packaging capacity of the vector will be reduced to approximately 3.

AAV2 was one of the first AAV serotypes identified and characterized, including the sequence of its genome. The sequences placed between the ITRs will typically include a mammalian promoter, gene of interest, and a terminator Fig. In many cases, strong, constitutively active promoters are desired for high-level expression of the gene of interest. All of these promoters provide constitutively active, high-level gene expression in most cell types. Some of these promoters are subject to silencing in certain cell types, therefore this consideration needs to be evaluated for each application [ 16 ].

Schematic representation of the basic components of a gene insert packaged inside recombinant AAV gene transfer vector. Although many therapeutic strategies involve systemic delivery, it is often desirable to have cell- or tissue-specific expression.

Likewise, for local delivery strategies, undesired systemic leakage of the AAV particle can result in transduction and expression of the gene of interest in unwanted cells or tissues. Likewise, the neuron-specific enolase promoter can attain high levels of neuron-specific expression [ 18 , 19 ].

Often is the case, systemic delivery of AAV results in a significant accumulation in the liver. While this may be desirable for some applications, AAV can also efficiently transduce other cells and tissues types. Finally, there are now technologies that have the ability to generate novel, tissue-specific promoters, based on DNA regulatory element libraries [ 22 ]. Over the course of the past 10—15 years, much work has been done to understand the correlation between codon usage and protein expression levels.

Although bacterial expression systems seem to be most affected by codon choice, there are now many examples of the effects of codon engineering on mammalian expression [ 23 ]. Many groups have developed their own codon optimization strategies, and there are many free services that can similarly provide support for codon choice. Codon usage has also been shown to contribute to tissue-specific expression, and play a role in the innate immune response to foreign DNA [ 24 , 25 ].

With regard to the gene of interest, codon engineering to support maximal, tissue-specific expression should be performed. Although there is much known about these individual components that needs to be considered when designing an AAV vector, the final design will most likely need to be determined empirically. It is not yet possible to know how a particular design will function by just combining the best elements together based on published reports, therefore considerable trial and error will eventually be required for deciding on the final construct.

In addition, one also needs to consider the differences between in vitro and in vivo activity. Although it is possible to model rAAV expression in rodents, there is still significant concern about the translatability to humans. AAV has evolved to enter cells through initial interactions with carbohydrates present on the surface of target cells, typically sialic acid, galactose and heparin sulfate [ 29 , 30 ]. Subtle differences in sugar-binding preferences, encoded in capsid sequence differences, can influence cell-type transduction preferences of the various AAV variants [ 31 — 33 ].

For example, AAV9 has a preference for primary cell binding through galactose as a result of unique amino acid differences in its capsid sequence [ 34 ]. It has been postulated that this preferential galactose binding could confer AAV9 with the unique ability to cross the blood—brain barrier BBB and infect cells of the CNS, including primary neurons [ 35 , 36 ].

In addition to the primary carbohydrate interactions, secondary receptors have been identified that also play a role in viral transduction and contribute to cell and tissue selectivity of viral variants. As a result of these subtle variations in primary and secondary receptor interactions for the various AAV variants, one can choose a variant that possesses a particular tropism and preferentially infects one cell or tissue type over others Table 1.

For example, AAV8 has been shown to effectively transduce and deliver genes to the liver of rodents and non-human primates, and is currently being explored in clinical trials to deliver genes for hemoglobinopathies and other diseases [ 38 ].

Likewise, AAV1 and AAV9 have been shown to be very effective at delivering genes to skeletal and cardiac muscle in various animal models [ 39 — 46 ].

Engineered AAV1 is currently being explored as the gene transfer factor in clinical trials for heart failure, and has been approved for the treatment of lipoprotein lipase deficiency [ 47 ].

However, although different AAV vectors have been identified that preferentially transduce many different cell types, there are still cell types for which AAV has proven difficult to transduce. With the strong desire to utilize AAV to deliver genes to very selective cell and tissue types, efforts to clone novel AAV variants from human and primate tissues have identified a number of unique capsid sequences that are now being studied for tropism specificities [ 48 ].

In addition, recombinant techniques involving capsid shuffling, directed evolution, and random peptide library insertions are being utilized to derive variants of known AAVs with unique attributes [ 49 — 51 ]. In vivo-directed evolution has been successfully used to identify novel AAV variants that preferentially transduce the retinal cells of the eye, as well as other cell populations, including those in the CNS [ 50 , 52 , 53 ].

In addition, these techniques have been employed to identify novel AAV variants with reduced sensitivities to neutralizing antibodies NAbs [ 54 — 57 ]. Alternatively, other investigators have inserted larger binding proteins into different regions of AAV capsid proteins to confer selectivity. For example, DARPins designed ankyrin repeat proteins , portions of protein A, and cytokines, have all been engineered into the capsid of AAV for the purpose of greater cell specificity and targeting [ 58 , 59 ].

As we continue to learn more about the biology of AAV with regard to the mechanisms involved in membrane translocation, endosomal escape, and nuclear entry, we will undoubtedly find opportunities to engineer unique properties into viral vectors through modulating one or more of these functions. For example, it has been hypothesized that surface-exposed serine and tyrosine residues could be phosphorylated upon viral cell entry, resulting in their ubiquitination and proteolytic degradation [ 62 — 64 ].

Studies have shown that mutation of tyrosine to phenylalanine, which prevents this phosphorylation, results in dramatically improved transduction efficiencies [ 63 ]. Similar efforts have been made in attempts to limit the effects of NAbs, as discussed below.

As one gives careful consideration to these selection criteria, it is possible to narrow the choices of which AAVs natural or engineered to profile. Alternatively, one can begin the path of exploring fully engineered versions of AAV for truly selective cell targeting and optimized transduction.

Because our understanding of AAV biology is in relative infancy, many of these efforts will remain empirical for quite some time as optimization for one activity could have a negative impact on another. Nonetheless, the future looks promising for this highly adaptable platform. One of the appealing aspects of using rAAV as a gene transfer vector is that it is composed of biomolecules, i.

Fortunately, a full-package virus lacks engineered lipids or other chemical components that could contribute to unwanted toxicities or immunogenicities that may not be predictable or fully understood.

In general, AAV has been shown to be less immunogenic than other viruses. Although not completely understood, one possible reason for this may hinge on the observation that certain AAVs do not efficiently transduce antigen-presenting cells APCs [ 65 ].

Additionally, unlike previous viral delivery strategies, rAAV does not contain any viral genes, therefore there will be no active viral gene expression to amplify the immune response [ 66 ]. Although AAV has been shown to be poorly immunogenic compared with other viruses i. This is further complicated by the fact that most people have already been exposed to AAV and have already developed an immune response against the particular variants to which they had previously been exposed, resulting in a pre-existing adaptive response.

It should be of no surprise that the formidable challenge is how to deliver a therapeutically efficacious dose of rAAV to a patient population that already contains a significant amount of circulating NAbs and immunological memory against the virus [ 67 ].

Whether administered locally or systemically, the virus will be seen as a foreign protein, hence the adaptive immune system will attempt to eliminate it. This leads to plasma cell and memory cell development that has the capacity to secrete antibodies to the AAV capsid. These antibodies can either be neutralizing, which has the potential to prevent subsequent AAV infection, or non-neutralizing. Non-NAbs are thought to opsonize the viral particles and facilitate their removal through the spleen [ 70 ].

Upon entry of the virus into target cells during the course of the natural infection process, the virus is internalized through clathrin-mediated uptake into endosomes [ 71 ]. After escape from the endosome, the virus is transported to the nucleus where the ITR-flanked transgene is uncoated from the capsid [ 72 ]. The pathway and mechanism of AAV intracellular transport and processing is not fully understood, and there are quite a few areas of debate with regard to current understanding.

The most current hypothesis is that following endosomal escape, capsid breakdown and uncoating occurs after subsequent nuclear translocation. However, it is thought that cytosolic ubiquitination of the intact virus can occur during transport to the nucleus [ 73 ]. This would be a critical step in directing capsid proteins to the proteasome for proteolytic processing into peptides for class I MHC presentation.

This hypothesis is supported by data in which proteasome inhibitors, or mutations in capsid residues that are sites for ubiquitination, can limit class I presentation and T-cell activation [ 73 — 76 ]. However, apparent differences have been observed for T-cell activation to different AAV variants with significant sequence identity. At this time, it is unclear whether this is due to subtle capsid sequence differences and susceptibility to MHC I presentation or differential cellular processing that is innate to the different AAV variants, or simply due to contaminants in vector preparations [ 76 ].

In addition to an adaptive immunological reaction to the capsid of AAV, the transgene can elicit both an adaptive and an innate response. If the transgene encodes a protein that can be recognized as foreign, it too can generate a similar B- and T-cell response. For example, in replacement therapy applications in which the protein to be replaced is the consequence of a null genotype, the immune system will have never selected against precursor B and T cells to that protein [ 70 , 77 ].

Likewise, if the transgene is an engineered variant, the engineered sequence can be recognized as foreign. Even the variable regions of antibodies can activate an adaptive response that can result in deletion of target cells that are expressing transgene as a result of AAV delivery.

Finally, a transgene with a significant number of CpG dinucleotides can activate innate responses through toll-like receptor TLR molecular pattern receptors [ 78 ]. To date, this represents one of the biggest therapeutic challenges to the use of systemically delivered AAV, and is thought to be one of the factors in early clinical failures [ 79 ].

Pre-existing immunity to AAV can often be overcome by selecting a particular AAV variant that has not circulated throughout the human population, and, therefore, does not have any memory responses elicited against it, including NAbs and T cells [ 80 ].

Additionally, some of the AAV evolution technologies discussed above have been used to identify AAVs that are resistant to the effects of NAbs [ 50 , 57 ]. Although not optimal, it is possible to prescreen subjects for the presence of NAbs to the particular AAV variant to be used.