Clinical aspects

Description

Gene transfer to human patients may be ex vivo or in vivo.

A simplified classification of various methods of gene therapy is shown in Table 2.

Table 2. A Simplified Classification of Gene Therapy Techniques

Ex vivo gene therapy. Ex vivo gene transfer techniques usually involve the genetic alterations of cells (cell lines or human cells), mostly by use of viral vectors, prior to implanting these into the tissues of the living body. These methods have been frequently used in clinical trials because they are usually more efficient than in vivo methods. One disadvantage is that reimplantation of genetically engineered cells grown in culture may not result in long-term survival of a large portion of the cells unless they are protected by encapsulation before injection.

In vivo gene therapy. In vivo gene therapy means direct introduction of genetic material into the human body. It can be accomplished by nonviral vectors. The advantages of direct in vivo methods are:

The disadvantages of in vivo methods are:

In vivo gene delivery may be local (in situ) or systemic. In situ gene therapy means the introduction of genetic material directly into a localized area in the human body. The site of gene delivery may be irrelevant in systemic delivery with some form of targeting, provided the therapeutic molecule is released effectively and can reach its final site of action. In vivo gene delivery is likely to combine specific targeting systems with novel transcription switches.

Vectors are modified to enhance delivery efficiency to target locations in the CNS. Several gene therapy approaches are approved by the FDA for CNS disease or injury treatment, which is due, in part, to the unpredictable nature of many vector systems, eg, safety concerns exist with the use of viral vectors for CNS gene delivery. Therefore, nonviral, biomaterial vectors are being explored for CNS application (47).

Various methods of gene transfer as applied to the nervous system are shown in Table 3.

Table 3. Methods of Gene Transfer as Applied to Neurologic Disorders

Viral vectors. Viral transduction involves infection of the cell with a modified virus and introduction of a viral genome containing the foreign genes. Although the concept of gene therapy and viral vectors is recent, vaccinations for diseases such as smallpox involved the transfer of "foreign" genetic material into patients. In viral vectors, the genes required for replication are removed and replaced by therapeutic genes and selection markers. Viral vectors are used not only as carriers of therapeutics but have facilitated development of genetic models closely replicating human CNS pathologies and have changed our concept of the molecular events leading to neuronal cell death. Important viral vectors that have been used as vectors for gene therapy are:

Retroviruses. Retroviral vectors, in the form of Moloney murine leukemia virus and its relatives, have one of the longest pedigrees in gene transfer therapy. The advantages of using retroviral vectors to insert human genes in virus-infected human cells in vitro conditions, is the high efficiency of gene transfer into dividing cells. This depends on the ability of the reverse-transcribed retroviral DNA to integrate into the chromosomal DNA. The high titer of the virion capable of infecting the target cells and lack of virus genes for virus replication and egress from the infected cells prevents virus replication. These are the most used vectors for gene therapy, but the limitation for application in the CNS is that retroviruses infect only mitotic cells, and there is a difficulty in transferring genes to postmitotic neurons. However, the ability to integrate selectively into dividing cells has made them excellent vectors for delivery of "killer" genes to the brain tumors.

Foamy viruses are nonpathogenic retroviruses that infect various animals including nonhuman primates and can be transmitted to humans through zoonotic infection. The replication strategy of Foamy viruses differs from that of other retroviruses, and they are safer than gammaretroviruses or lentiviruses. They can mediate efficient and stable gene transfer to hematopoietic stem cells. Several other unique properties of Foamy virus vectors make them well suited for therapeutic gene transfer, including a broad tropism, a large transgene capacity, and the ability to persist in quiescent cells. Foamy viruses have been successfully used for in vivo gene therapy in the X-linked severe combined immunodeficiency canine model (48).

Adenoviruses. Recombinant adenoviruses have gained widespread popularity for gene transfer. Adenoviral vectors for gene therapy are adenoviruses that have been genetically modified by introducing deletions in the viral genome to create space for a foreign gene to be inserted, and to create a replication defective virus. Adenoviruses can be used to infect nondividing cells. Adenovirus genomes usually do not integrate into the host cell chromosomes, and are suitable for directing transient expression. Replication-defective adenoviral vectors with deletions in certain regions have large space for carrying foreign genetic material and are suitable for treatment of neurologic disorders.

Adeno-associated viruses. These viruses are associated with any known human disease and can be used to infect postmitotic neurons. In a productive life cycle, adeno-associated viruses require coinfection with helper viruses (adenovirus, herpes virus, or vaccinia virus) for replication. These viruses are rapidly and selectively taken up in the brain and would facilitate targeted brain gene therapy. In vivo somatic gene transfer using adeno-associated virus has the advantage of being neuronal gene expression efficient, long-lived, and nontoxic. rAAV4gal shows a distinct affinity for transduction of the ependyma, which is a desirable feature when attempting to restrict gene delivery to ependymal cells. Secretion of a transgene product from transduced ependymal cells into the cerebrospinal fluid could be a useful method of protein delivery. rAAV4 has the potential to transfer genes to stem cells in the ependyma, which could lead to transgene expression in differentiated populations of progeny cells. This characteristic combined with persistent gene expression could reduce the need for multiple injection sites and repeat injections. Other advantages of adeno-associated virus vector include the lack of any associated disease with a wild-type virus, the ability to transduce nondividing cells, the possible integration of the gene into the host genome, and the long-term expression of transgenes. The development of novel therapeutic strategies for neurologic disorders by using AAV vector has an increasing impact on gene therapy research. AAV vectors support localized long-term, gene expression in the central nervous system. More than 20 clinical trials have been done to prove therapeutic effect of AAV vectors on neurodegenerative diseases, retinopathies, and neuromuscular diseases (45). AAV9 is efficient when directly injected into the brain, but it also produces global expression in the brain and spinal cord after a peripheral, systemic route of administration in mice (14). A method that can affect the entire CNS without injecting the CNS is promising, not only for basic functional experiments but also for clinical gene therapy. Safety and tolerability of rAAV delivery in the human brain at moderate doses is established, but therapeutic effects are modest. With further developments, AAV vectors are becoming an efficient therapy platform for the treatment of neurologic diseases. Importantly, initial clinical studies have demonstrated encouraging safety and efficacy in diseases such as Parkinson disease and spinal muscular atrophy, as well as durability of transgene expression.

Herpes simplex virus. Herpes simplex virus-1 is a neurotropic virus. Following infection at the periphery, the virus spreads preferentially in the nervous system. Of all the viral vectors, herpes simplex virus is most relevant to neurologic disorders. Herpes simplex virus-1 amplicon vector is a plasmid-based vector that has been successfully used to transduce and express various genes in somatic cells, particularly postmitotic neurons. Replication-defective herpes simplex virus vectors, which efficiently transfer and express the cDNA for fibroblast-growth-factor-2 in vitro and in vivo, are suitable for studies of fibroblast-growth-factor-2 involvement in neurologic disorders. Herpes simplex virus amplicon, administered by stereotactic injection into the striatum in experimental animals, induced a rather modest interferon response, and transgene expression lasted more than 1 year despite dose-dependent inflammation and infiltration of immune cells around injection sites (52). Such vectors have potential for gene therapy of chronic neurologic disorders.

Herpes simplex virus-1 vectors have been used to introduce enzymes of inherited metabolic disorders, neurotransmitter synthesizing enzymes, neuropeptides, signal transduction enzymes, glutamate receptors, and neurotrophic factors. The development of herpes simplex virus-1 defective virus vectors for delivering foreign genes in vitro and in vivo has the potential to significantly enhance both the understanding of the genetic basis of neuronal function and our ability to treat a range of neurologic disorders. The natural axonal transport mechanism of herpes simplex virus-1 is exploited, enabling transgene expression in the cell nucleus within the inaccessible trigeminal ganglion or dorsal root ganglion, following the noninvasive procedure of subcutaneous vector inoculation (22). Potential applications of herpes simplex virus vectors in neurology are as follows:

Lentiviral vectors. They can transduce postmitotic cells and show strong neural tropism. Lentiviral vectors in which the expression of the transgene can be regulated are now available. Major applications of recombinant lentiviral vectors have been in polyglutamine disorders, such as synucleinopathies and Parkinson disease, or in investigating gene function in Huntington disease, dystonia, or muscular dystrophy (18).

Semliki Forest virus vectors. These vectors can infect numerous mammalian cell lines and primary cell cultures; they can result in high levels of transgene expression in hippocampal slices, as well as in vivo in rodent brain.

Nonviral vectors. Due to some concern with the use of viruses as vectors, a search has been made for alternative methods of gene therapy. Nonviral methods rely on cellular mechanisms to import DNA into the cell and to transport the DNA to the nucleus. The simplest method would be to administer functional plasmid DNA directly to tissues using methods used for other drug molecules. Most of the biologics are given by injection and this method has been tried for gene therapy as well. DNA can be delivered to the tissues "naked" or conjugated with carriers such as liposomes. The following are some of the nonviral methods of gene delivery.

Naked DNA-mediated gene transfer. This was first demonstrated in the skeletal muscle, but other organs have been shown to share this characteristic. Naked DNA injected intracerebrally into the adult mouse brain can provide expression of luciferase, a reporter protein. Naked DNA is as efficient in the adult brain as in the newborn.

Liposomes for gene therapy. The successful encapsulation of whole virus or DNA into liposome opened exciting possibilities by enhancing DNA introduction into mammalian cells, which is a sort of chemical transfection. The lipid coating enables the cells to survive after injection and deliver DNA by fusion with the cell. Conventional DNA-encapsulating liposomes are unpopular as carriers because of the specialized expertise required to prepare these liposomes, in addition to custom DNA loading for every type of cell used as a target. The procedure was cumbersome and inefficient, especially in encapsulation of larger fragments. The technique was applicable mostly in vitro and its use rapidly declined. Another candidate for DNA delivery was the virosome, a liposome containing fusogenic viral proteins. The problem with such structures is effective DNA encapsulation. Because these structures contain viral proteins, immunogenicity can be a problem. The use of liposomes to deliver a gene in vivo with the aim of inducing the production of encoded protein was revived with emergence of cationic liposomes.

In the DNA-cationic liposome complex, the nucleic acid or short, single-strand antisense oligonucleotide is not encapsulated but is simply covered with small unilateral vesicles by electrostatic interactions. Cationic liposome formulations for gene transfer have also overcome other problems associated with liposome-mediated gene transfer. Cationic liposome-DNA complexes are positively charged and bind easily to the negatively charged cell surface with highly efficient transfection. DNA is eventually released in the cytoplasm.

DNA-liposome complexes enhance DNA delivery in the newborn brain but not in the adult. This is due to the lowered ability of the adult brain to be transfected by cationic lipid preparations rather than due to any variation in uptake and expression of naked DNA. The dividing and migrating cells in the newborn are likely more susceptible to liposome-based gene transfer. Limited diffusion or bioavailability of these complexes with aging may be due to increased myelination.

Trojan Horse Liposome technology, using conjugates of monoclonal antibodies that bind to specific endogenous receptors, has been applied to nonviral gene therapy of brain disorders as a solution to the delivery obstacle across the blood-brain barrier (09). This facilitates receptor-mediated transcytosis of the conjugate through the blood-brain barrier and endocytosis into brain cells. This technique has potential applications in the gene therapy of Parkinson disease and brain tumors.

Systemic administration of a targeted liposomal synthetic vector, containing a therapeutic DNA under the control of an inducible promoter, could provide an efficient tool for gene therapy of CNS disorders. Intravenous gene transfer using pegylated immunoliposomes targets the plasmid DNA to brain with receptor-specific transport ligands, which act as a molecular Trojan horse to ferry the gene across both the blood-brain barrier and the neuronal cell membrane. By use of this approach, striatal tyrosine hydroxylase activity is completely restored in the 6-hydroxy-dopamine model of experimental Parkinson disease (61).

Ligand-polylysine-DNA complexes. Lipopolyamines have been reported to be efficient transfection reagents, as they do not require any co-lipid for activity. Cationic polyamine is the essential part of the reagent, and it complexes with the anionic DNA. Another preparation, poly-L-lysine has the advantage that different ligands can be coupled with its primary amino groups. Poly-D-lysine has been converted into a phospholipid derivative, which has a level of transfection equal to that of phospholipid derivative and disease-oriented physician education liposomes, indicating that degradation of polylysine chain, which likely takes place in the lysosomes, is not necessary for the cytoplasmic delivery of DNA. The gene delivery in this case may be receptor-mediated. These have also been used for targeted gene delivery.

Polyethylenimine has been shown to provide exceptionally high levels of transgene expression in the mouse brain. This vector has a potential for gene therapy of brain disorders.

Receptor-mediated endocytosis. To circumvent the limitations of direct introduction of DNA by nonviral methods, techniques have been developed to deliver DNA via a receptor-mediated pathway. These vectors, called molecular conjugate vectors, use the internalization mechanism intrinsic to specific macromolecules. This involves the targeting of genes to specific cell-surface receptors such as transferrin receptors. DNA is linked to a targeting molecule such as polylysine and the conjugate-DNA complex would then bind to a specific cell surface receptor, inducing endocytosis and the transfer of DNA into the cells.

Human artificial chromosomes. Long synthetic arrays of alpha satellite DNA are combined with genomic DNA to generate artificial chromosomes. The resulting linear chromosomes contain exogenous alpha satellite DNA and contain all the sequence elements required for stable mitotic chromosome segregation and maintenance. There is hope that functional human artificial chromosomes will serve as valuable nonviral gene transfer vectors. To utilize human artificial chromosomes for human gene therapy, efficient nontoxic methods for delivery to somatic cells in vivo will be required. Another requirement for artificial chromosomes is that they can replicate. Sequences that can mediate replication initiation are frequent in human DNA, so it is likely that no special provision for replication is necessary in human artificial chromosomes. To maintain a linear conformation the chromosome ends must be capped with human telomeres.

Nanoparticles. Nanoparticles can be used as nonviral vectors for efficient in vivo gene delivery without significant toxic effects and with efficacy equaling that of viral vectors. Synthetic nanodelivery systems have formulations and structures that mimic viruses but retain the safety elements of synthetic, nonviral systems (28). Nanocarriers such as liposomes, metallic and polymeric nanoparticles, dendrimers, gelatins, and quantum dots/rods have been developed, and each shows distinct characteristics (58). Pharmacokinetics and possible neurotoxicity of a nanocarrier should be assessed when selecting an appropriate vector for gene therapy.

Ultrasound-mediated gene delivery. Ultrasound contrast agents are well-known for diagnosis and use of ultrasound is now being developed for delivery of drugs as well as genes because of triggered release at the target while sparing surrounding healthy tissue, which has certain advantages for treatment of neurologic disorders. Although promising results have been reported in preclinical studies on animal models, there are still only few examples of clinical use (06).

Ideal vector for gene therapy of neurologic disorders. Viral vectors continue to play an important role in genetic manipulations of neuronal and non-neuronal cells. Viral vectors are expensive. Other drawbacks include severe side effects such as immunogenicity and carcinogenicity, poor target cell specificity, and their inability to transfer large-sized genes because of limited capacity. Therefore, nonviral vectors are preferred.

The relative inaccessibility of the brain also presents a problem with systemic administration of a vector that may not cross the blood-brain barrier. The ideal neuronal gene transfer vector should have the following main characteristics:

Promoters of gene transfer. An ideal promoter of a therapeutic gene should be active for a long term and it should be tissue-specific and even cell-specific. Viral promoters satisfy the former criterion but not the latter. Promoters specific for the nervous system should be used to drive transgenes and should include the promoters for the following:

The ability to maintain long-term gene expression in specific cell types within the brain is a fundamental requirement for CNS gene therapy. Non-integrating vectors such as adenoviral vectors can be used to mediate powerful, long-term episomal transgene expression in neurons provided that a powerful neuron-specific viral post-transcriptional regulatory element vector cassette is used at titers that do not elicit an immune response. In situations where the quantity of a transgene is crucial, the use of an externally regulated (inducible) promoter or enhancer unit might be necessary.

Gene editing. CRISPR (clustered regularly interspaced short palindromic repeats) can be used for precisely altering the genomes of living cells by adding or deleting genes (13). To create this technique, a set of bacterial proteins that normally defend against viral invaders were modified. CRISPRs are used by bacteria to help store short genetic codes of viruses that have previously invaded them, and as new foreign DNA molecules are encountered, they are identified using these genetic records. Making use of naturally occurring bacterial protein-RNA systems, which recognize and snip viral DNA, enables creation of DNA-editing complexes that include a nuclease called Cas9 bound to short RNA sequences. These sequences are designed to target specific locations in the genome; when they encounter a match, Cas9 cuts the DNA. By triggering a conformational rearrangement in Cas9, the guide RNA determines target DNA binding (38). Cas9 nucleases can be directed by short RNAs to induce site-specific DNA cleavage at endogenous genomic loci in human cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. A nano-micelles formulation created to contain the Cas-9-encoding mRNA and the gRNA, surrounding them with separate shells of polyethylene glycol for rapid release and degradation, was injected into the brains of transgenic mice to achieve a higher level of genome editing efficiency, which brings the use of CRISPR as a therapeutic closer to becoming a practical reality (01).

CRISPR/Cas9 has potential clinical applications, eg, treatment of progressive blindness caused by dominant forms of retinitis pigmentosa by inactivating the mutant allele in retinal cells. Due to consequence of inherited or de novo channelopathies, which result from loss or gain of mutations in genes coding ion channels, the brain may develop and wire abnormally, and the choice of optimal gene therapy or gene editing strategy will depend on the time of intervention (germline, neonatal, or adult). Advances in gene editing technologies may lead to novel treatments for channelopathies by repairing disease-causing channel mutations at the germline level (57).

Gene editing of neural stem cells enhances their gene targeting ability, and they have the potential for cell/gene therapy of neurodegenerative disorders and lysosomal storage disorders (15).

Analysis of the views of over 1000 researchers in gene editing from all over the world shows that, in the next decade, DNA double-strand breaks are expected to be the main method for gene editing, and CRISPR-Cas systems will become the mainstream programmable nuclease with applications in the treatment and prevention of a variety of human diseases (50).

RNA trans-splicing. RNA trans-splicing is the joining of 2 different pre-mRNA molecules from the same gene to form a composite mRNA. This could enable a mutated region of pre-mRNA responsible for disease to be excised and replaced with a normal protein-coding sequence. RNA trans-splicing could provide the basis of therapeutic strategies for correcting impaired alternative splicing caused by pathogenic mutations in cis-acting splicing elements.

Antisense therapy. Antisense approach to gene therapy involves the use of substances that intervene in the natural processing of genetic information in the cell, particularly in the case of a disease due to genetic aberration. The basic mechanism is to block the synthesis of cellular proteins by interfering with either the transcription of DNA to mRNA, or the translation of mRNA to proteins.

The translation arrest can be induced endogenously by plasmid-derived antisense mRNA transfected via a suitable vector, or exogenously by an antisense oligonucleotide. Various subclasses of antisense agents (antisense sequences, antigene sequences, and ribozymes) interrupt the process of transcription or translation at different points.

An oligodeoxynucleotide is a synthetic piece of DNA composed of a chain of smaller units called nucleotides, and is complementary to RNA or DNA of the genome, or a RNA derived from it. All oligodeoxynucleotides should contain at least 15 nucleotides. These compounds prevent or disable the expression of disease-associated proteins. Ribozymes catalyze RNA cleavage. High affinity RNA ligands are termed "decoys" or "aptamers." Triplex approach, which involves the attachment of oligonucleotides to chosen segments of DNA is also included in this approach, although, strictly speaking, triplex sequences are not antisense.

Antisense therapy is a form of gene therapy because it is a modulation of gene function for therapeutic purposes. Some consider antisense therapy to be the "flip" side of gene therapy, in which a good gene is inserted to produce health-giving proteins. Antisense drugs, on the other hand, block the production of disease-causing proteins. Oligonucleotides differ from standard gene therapies because they cannot give rise to proteins but can only block the expression of existing genes. Most gene therapies provide full healthy genes as substitutes for versions that are missing or unable to direct adequate synthesis of needed proteins. Use of antisense oligodeoxynucleotides has been explored in neurodegenerative and cerebrovascular disorders.

RNA interference and gene therapy. RNA interference is a cellular mechanism to regulate the expression of genes and the replication of viruses. RNA interference or gene silencing involves the use of a double-stranded RNA. Once in the cell, the double-stranded RNAs are processed into short 21 nucleotide to 22 nucleotide double-stranded RNAs, termed small interfering RNAs (siRNAs), which are used in a sequence-specific manner to recognize and destroy complementary RNAs.

RNA interference (RNAi) has some similarities to the antisense approach, but it involves gene silencing rather than gene knockout achieved by antisense oligonucleotides. Because of their high specificity and efficiency, small interfering RNAs might be a new class of antigene medicines for gene therapy applications. Research is in progress to find a treatment for amyotrophic lateral sclerosis using RNAi. Diseases such as fragile X syndrome, which is caused by expansion of trinucleotide repeats, can be targeted by RNAi. Polyglutamine toxicity is suppressed by RNAi, which targets the expanded mRNA transcript. Viral as well as nonviral vectors can be used for efficient delivery of RNAi. RNAi has great potential in becoming a successful therapeutic strategy for spinocerebellar ataxia and Huntington disease where involved genes have been identified and can be specifically silenced. Use of siRNA to treat several CNS disorders is limited by delivery of viral as well as nonviral vectors to specific locations in the brain mainly because of the blood-brain barrier (25). Innovative techniques are under investigation for improving targeted delivery of siRNAs.

Routes of delivery of genes to the CNS. In preclinical studies, transgenes encoding therapeutic proteins, microRNAs, antibodies, or gene editing machinery have been successfully delivered to the central nervous system with natural or engineered viral capsids via various routes of administration (16). The following routes have been used:

Direct injection into CNS. Development of stereotactic techniques in neurosurgery has enabled injection of genes and vectors into specific locations in the brain both in experimental animals and humans. These techniques evolved for creating lesions to modify disturbances of function, as well as for administration of therapeutic substances such as cell grafts and neurotrophic factors. These techniques still carry some risk and some areas of the brain are still considered inaccessible by these approaches.

Injection in specific locations. Both herpes simplex virus and adenoviral vectors have been considered suitable for direct injection into the brain. Retroviruses are considered only for injection into brain tumors. Injection of naked DNA or a vector can be made in a specific location. However, it is uncertain if the injected material would remain confined to the specific location. The injected material may diffuse via neural or CSF pathways to other areas of the brain. Another problem is the limited duration of effect and difficulty in repeating injections in the same location.

Direct stereotactic injection vector into the pituitary of a recombinant adenoviral vector carrying pituitary gland-specific promoters may be useful for targeted gene therapy of pituitary diseases.

Introduction of the genes into cerebral circulation. Vascular delivery of viral vectors would optimize delivery to the CNS without the need for multiple invasive procedures. Most of the efforts to infect neurons and neuroglia after vascular delivery have been unsuccessful, presumably because of the inability of the viral particles to cross the blood-brain barrier. Osmotic disruption of the blood-brain barrier has been used for the delivery of chemotherapeutic agents and proteins to the brain. Ultraviolet-inactivated noninfectious herpes simplex virus particles have been delivered via blood-brain barrier disruption in rats.

Functional transgene expression in a rat brain has been reported after intracarotid injection of recombinant adenovirus and intracarotid infusion of mannitol to transiently disrupt the blood-brain barrier.

Introduction of genes into cerebrospinal fluid. Drugs can be introduced directly into the CSF either by lumbar puncture, cisternal puncture (at the craniospinal junction), or into the lateral ventricles of the brain. The last route is more complicated because it involves traversing the brain substance, and repeated punctures can damage the brain. One way to reduce this trauma is to insert a cannula connected to a subcutaneous reservoir (Ommaya reservoir). The drugs or other agents can then be introduced by an injection into the reservoir through the skin. Therapeutic recombinant proteins can be introduced into the brain via this route. Glial cell line-derived neurotrophic factor has been used for the treatment of Parkinson disease.

Delivery of AAVs into the CSF is an efficient approach to target the CNS and bypass the blood-brain barrier. An experimental study has shown that lumbar intrathecal delivery of single stranded AAV9 is a safe and highly efficient means of targeting the CNS in adult mice (07). Studies in larger animals have been done to find the optimal route for administration into humans. One study has compared intracerebroventricular and intracisternal AAV administration in dogs, and found that both routes resulted in similarly efficient transduction throughout the brain and spinal cord, but intracerebroventricular injection produced encephalitis associated with a T-cell response to the transgene product (30). An extension of this study evaluated vector administration via lumbar puncture in nonhuman primates, with some animals placed in the Trendelenburg position after injection to facilitate entry into the brain, which did not improve transfer.

Intravenous administration of vectors. Tissue-specific gene expression in brain is possible after intravenous administration of a nonviral vector with the combined use of gene targeting technology and tissue-specific gene promoter.

Ependymal-leptomeningeal route can be used for the injection of nonreplicative vectors into the cerebrospinal fluid space. These vectors can infect the ependymal and leptomeningeal cells consistently and without side effects, and in turn, produce the 'therapeutic' product of the transgene in the CNS for extended periods of time. This approach would be useful in gene therapy of multiple sclerosis.

Implantation of genetically engineered encapsulated cells producing ciliary neurotrophic factor in the spinal subarachnoid space is an example of gene delivery into the CNS via this route. Deposition of transgenic constructs into the subarachnoid space or the ventricular system is likely to be an efficient route for transducing not only the subependymal region but also for dissemination of products of gene expression into the brain. Intracerebroventricular or intrathecal injection of cationic liposome: DNA complexes can produce significant levels of therapeutically relevant gene expression within the CNS.

Introduction of antisense compounds into the CSF pathways. Antisense therapy is aimed at suppression of detrimental agents or function in the CNS. Antisense oligodeoxynucleotides can penetrate the CNS tissue and enter both astrocytes and neurons. Regional administration of oligodeoxynucleotides into the CSF has been explored. There is little intrinsic nuclease activity in the CSF to degrade oligodeoxynucleotides. Phosphorothioate oligodeoxynucleotides are resistant to degradation in the CNS, and after intraventricular administration they are cleared in a manner consistent with CSF bulk flow. Continuous infusion of oligodeoxynucleotides by a mini-osmotic pump in experimental animals can maintain micromolar concentrations of intact phosphorothioate oligodeoxynucleotides in CSF for longer periods without obvious neurologic or systemic toxicity. Animal experiments have shown the following effects of oligodeoxynucleotides administered into the cerebral ventricles:

Delivery of gene therapy to the peripheral nervous system. Targeted expression of foreign genes to the peripheral nervous system has many potential applications including gene therapy of neuromuscular diseases. Replication-defective viral vectors can be injected into dorsal root ganglia by microneurosurgical techniques.

Cell-mediated gene therapy. One of the gene transfer methods currently being explored is cell-mediated ex vivo gene therapy. This technique involves the genetic manipulation of cell, followed by their in vitro amplification and subsequent injection into target tissues. For human gene therapy, success of cell-mediated methods depends on the capacity of cells to proliferate during the stage of in vitro amplification. One of the limitations is the phenomenon of senescence of the diploid cells after several divisions. Several types of cells have been used for cell-mediated gene therapy: fibroblasts, myoblasts, keratinocytes, hepatocytes, neural cells, etc. Fibroblasts are the most used cells. Genetic modification of stem cells has been proposed as a treatment strategy for a variety of diseases. Autologous pluripotent stem cells, mesenchymal stem cells, and neural stem cells are most often used. The synergy between regenerative effects of stem cells and additional properties by engineering can provide significant benefits for neurodegenerative changes in the brain. Ex vivo gene therapy is being investigated in preclinical animal studies and clinical trials in humans to treat neurologic disorders with a focus on Parkinson disease, Huntington disease, Alzheimer disease, amyotrophic lateral sclerosis, and stroke (26).

Fibroblasts. Skin fibroblasts represent an attractive target for gene transfer from which soluble factors can be secreted into the serum. After in vitro retrovirus-mediated gene transfer, genetically modified skin fibroblasts can be reimplanted into a newly formed mesenchymal tissue by embedding them into collagen fibers. This approach has also been evaluated as a model for the treatment of lysosomal storage disorders. A clinical trial has been proposed in which neo-organs containing autologous fibroblasts genetically modified to secrete human alpha-L-iduronidase will be implanted in children with the severe form of Hurler disease.

Genetically engineered fibroblasts are "biological factories" for the production of neurotrophic factors such as basic fibroblast growth factor, brain-derived neurotrophic factor, and neurotrophin-3. This technique allows autologous grafting and induces neuronal survival and axonal regeneration in several animal models. One risk of this procedure is that displaced fibroblasts from improperly performed taps can produce epidermoid tumors, which would be a limiting factor for transplantation into the brain.

Stem cells. Neural stem cells may present an ideal route for gene therapy in addition to offering new possibilities for the replacement of neurons lost to injury or disease. A major obstacle to the clinical development of stem cell gene therapy rests in the inability to deliver a gene into a therapeutically relevant number of stem cells. For example, a retroviral vector system is usually unable to deliver genes to nondividing stem cells. However, recombinant lentiviral vectors and Foam virus vectors have shown the potential for further development of ex vivo gene therapy protocols for the brain. Replacement of the endogenous brain microglia from patients with X-linked adrenoleukodystrophy by autotransplantation of genetically corrected hematopoietic stem cells using a lentiviral vector is feasible

Neural stem cells propagated in culture can be reimplanted into mammalian brain where they might integrate appropriately throughout the mammalian CNS and stably express foreign genes. Neural stem cells might aid in reconstructing of maldeveloped or damaged CNS at the molecular and cellular levels.

Neuronal cells. Primary neurons have not been used much as transgene carriers because they are difficult to obtain, maintain in culture, and transfect by classical methods. An alternative is to stimulate the proliferation of cultured precursors of neurons by growth factors, thus, enabling gene transfer by classical methods. The advantage of using engineered neurons is that they can establish synaptic contact with host tissue and provide the necessary gene product.

One of the most widely used replication techniques for gene transfer into neuronal cells involves the use of retroviral vectors. These vectors can deliver the genes into neurons, both in culture and in vivo, without undergoing replication themselves. A retrovirus containing sequences encoding the shorter form of pro-beta-nerve growth factor messenger RNA under control of the long terminal repeat has been constructed enabling secretion of nerve growth factor through regulated pathways. Herpes simplex virus-1 also holds promise for gene delivery into neurons and has the capability to achieve stable and long-term (2 to 3 weeks) expression of foreign genes into the nervous system. Investigations are in progress to determine the ideal site for insertion of the nerve growth factor gene into the herpes simplex virus-1 genome and a suitable promoter to achieve expression during the latent stage. Adenoviral vectors have been used for gene transfer in primary neuron cultures.

Astrocytes. Astrocytes are known to provide support to neurons by secreting molecules that promote neuronal differentiation, survival, and regeneration. Astrocytes have been engineered to secrete nerve growth factor or brain-derived neurotrophic factor. Genetically modified astrocytes producing nerve growth factor promote the prolonged survival and function of co-grafted adrenal chromaffin cells in a rat model of Parkinson disease. Using replication-defective adenoviral vectors, astrocytes can be efficiently engineered to secrete bioactive ciliary neurotrophic factor or neurotrophin-3, resulting in enhanced survival of responsive peripheral and central neuronal populations. Genetically engineered astrocytes appear to be useful and promising transgene carriers for CNS gene therapy.

Cerebral endothelial cells. Vascular endothelial cells are efficiently used as gene carriers in the peripheral organs. Brain endothelial cells are considered for gene transfer to the CNS. Compared with peripheral endothelial cells, brain endothelial cells display a unique phenotype characterized by presence of intercellular tight junctions and, along with the astroglia, constitute the blood-brain barrier. Immortalized cerebral endothelial cell lines have been used to deliver gene products to the rat brain. Survival of the grafted cells was observed for at least 1 year without any sign of tumor formation. When genetically modified to express beta-galactosidase and transplanted into the striatum, these cells were shown to integrate into the brain parenchyma and microvasculature. These cells have been engineered to secrete nerve growth factor and induce the formation of a dense network of low-affinity nerve growth factor receptor-expressing fibers near the implantation sites. This study establishes the cerebral endothelial cell as an efficient vector for gene transfer to the CNS.

Implantation of genetically engineered cells. Genetically engineered cells may be implanted for systemic delivery of recombinant proteins. This is one step further in the treatment of some acquired and genetic disorders by injections of recombinant proteins and is a type of gene therapy. A standard cell line engineered to secrete a desired gene product may be implanted at the appropriate site in the allogenic recipients. The selection of the site is influenced by several factors, which include the target organs or tissues in the case of local delivery of the therapeutic substance.

Implantation of genetically engineered cells is an ideal method for the delivery of neurotrophic factors across the blood-brain barrier. Engineered and encapsulated baby hamster cells have been used previously for intrathecal implantation in patients with amyotrophic lateral sclerosis, but the duration of delivery is limited and the dividing cells present the risk of overgrowth. Polymer encapsulated differentiated C2C12 myoblasts, when implanted into the intrathecal space in rats as a source of ciliary neurotrophic nerve factor, survive for long periods. Inclusion of herpes simplex virus-thymidine kinase in the expression vector provides a means of eliminating the dividing myoblasts by exposure to ganciclovir and increases the safety of this method.

Implantation of encapsulated cells. Encapsulation techniques involve the surrounding of the cells with selectively permeable membranes. The pores of the membranes should be small to block entry of immune mediators but large enough to allow inward diffusion of oxygen and nutrients required for cell survival and for outward diffusion of neuroactive molecules produced by the cells. Biohybrid implants represent a new class of medical device in which living cells, supported by hydrogel matrix and surrounded by a semipermeable membrane, produce and deliver therapeutic reagents to specific sites within a host. Nonautologous cells encapsulated in alginate microcapsule have also been used to deliver recombinate gene products. This strategy is being applied to the treatment of neurodegenerative disorders by neurotrophic factors. Encapsulation avoids some of the complications of free cell transplants including local reaction at the site of transplantation and tumor formation. Advantages of encapsulated cell-based therapy are:

Even though encapsulation makes cell-based therapies safer and more effective, there are still some disadvantages as follows:

Incorporation of nerve growth factor into polymers and sustained release of nerve growth factor from biodegradable polymer microspheres implanted intracerebrally has been proposed as a method of delivery of proteins and other macromolecules over a long period. From a neurosurgical point of view, stereotactic implantation of encapsulated biodegradable microspheres loaded with bioactive drugs is the ideal method of drug delivery. In the case of neurotrophic factors, this method can be applied to the following situations:

Targeted gene therapy. The ability to target gene transfer to a specific class of cells is an important aspect of improving efficacy of gene therapy and limiting undesirable side effects. Ideal gene therapy vectors would be delivered intravenously to transfect only specific cells. Existing vectors only transfect cells in vivo in a manner determined by blood flow and the site of introduction.

Gene transfer for neuromodulation. Neuromodulation is used for restoring neural function in disorders due to an imbalance in the activity of specific neural networks that is refractory to pharmacotherapy. Kir2.1, a gene for potassium channel, helps stabilize the resting potential of voltage-gated sodium channels to keep it below the threshold of activation. Therefore, the delivery of the Kir2.1 gene to neuronal cells could inhibit excessive neural activity. Kir2.1 gene has been delivered into the lumbar spinal cord of rats by an adenoviral vector and controlled by a gene switch system to induce its expression (10). This technique has potential for clinical applications.

Controlled induction of gene expression. Several inducible gene expression systems have been developed over the past decade to meet the need for regulated gene expression. Several methods that use elements within the vector design have been developed to enable an external drug or pro-drug to alter ongoing protein expression after in vivo gene transfer. The most promising and most studied regulated protein expression methods for in vivo gene transfer have been reviewed by Manfredsson and colleagues (41). Various methods of controlling gene or protein expression are shown in Table 4.

Table 4. Approaches to Controlling in Vivo Gene Expression in Gene Therapy

Monitoring of gene therapy. PET imaging enables imaging of target molecules in vivo, and numerous tracers are available for imaging of reporter gene activity. Selection of radiolabeled substrates that interact with specific transgene proteins has identified several reporter genes that can be used for imaging vector-mediated gene delivery and expression in both preclinical and clinical situations (12). One limitation of reporter gene systems is that they have not allowed monitoring of all areas of the brain. A PET reporter gene/probe system, which is capable of crossing the blood-brain barrier and targeting the pyruvate kinase M2 protein in the central nervous system with minimal endogenous expression in the brain, enables noninvasive monitoring of the level and location of gene expression in all areas of the brain, giving the medical team an early indication of the likelihood of treatment success. The proof of principle studies emphasize the superiority of [18F]DASA-23 to [18F]FDG in detecting the glycolytic response of glioblastoma to anticancer therapies in cell culture (05). A clinical trial evaluating the diagnostic utility of radiotracer [18F]DASA-23 PET in glioblastoma patients is ongoing for the early detection of therapeutic response to gene therapy in glioblastoma (NCT03539731). The aims of gene therapy for neurologic disorders are to provide a rational therapy by:

Indications

Examples of potential indications for gene therapy of neurologic disorders are shown in Table 5. Gene therapy of neurogenetic disorders, gene therapy of neurodegenerative diseases, gene therapy of muscular dystrophy, gene therapy of cerebrovascular diseases, and gene therapy of glioblastoma multiforme are described in detail in separate clinical summaries.

Table 5. Examples of Potential Indications for Gene Therapy of Neurologic Disorders

Separate clinical summaries in MedLink Neurology deal with applications of gene therapy in stroke, glioblastoma, neurogenetic disorders, neurodegenerative disorders, and muscular dystrophy.

Gene therapy for traumatic brain injury. Neural stem cells have been retrovirally transduced to produce nerve growth factor and transplanted into the injured brain with marked improvement of cognitive and neuromotor function and rescue of hippocampal neurons during the acute posttraumatic period. Brains with pial strip of the forelimb motor cortex as models for traumatic brain injury showed higher neuronal survival when treated with an adenovirus encoding VEGF zinc finger proteins (17). The contralateral forelimb function was also enhanced within the first 2 weeks after injury, indicating that VEGF ZFP therapy is neuroprotective following traumatic brain injury.

Gene therapy of epilepsy. Drug-resistant epilepsy is a problem in management, and even if seizures are controlled by drugs, there is risk of exposure of normal brain to the drugs and possible toxic effects that may lead to cognitive impairment. The ability of viral vectors to deliver therapeutics locally to seizure foci has the potential to overcome several limitations of medical therapy.

Recombinant adeno-associated viral vector-mediated delivery of neuropeptide Y and galanin genes was shown to have an inhibitory effect on seizures in preclinical studies. In a rodent model of epilepsy, optogenetic inhibition of a subset of principal neurons transduced with halorhodopsin targeted to the epileptic focus by lentiviral delivery was effective for attenuating electroencephalographic seizures (56). Local overexpression of the potassium channel Kv1.1 by lentiviral vector reduced the intrinsic excitability of transduced pyramidal neurons. Whether the same can be achieved by use of adeno-associated viral vectors that are commonly used in gene therapy trials is unknown. The lack of a nonhuman primate model of chronic recurrent seizures limits the extent to which gene therapy experiments can be translated to the treatment of human epilepsy, but there is potential for further development of these studies (62).

Gene therapy for peripheral nerve injuries. Gene therapy can be used to enhance the results of peripheral nerve repair as follows:

Gene therapy for intervertebral disc degeneration. Inflammatory cytokines in the intervertebral disc, particularly TNF-α and IL-1β, play a role in progression of degenerative disc disease, but current treatments do not target this mechanism. A study has used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) epigenome editing-based therapy for degenerative disc disease by modulating TNFR1/IL1R1 signaling in pathological human intervertebral disc cells (19). This downregulation significantly attenuated deleterious TNFR1 signaling but not IL1R1 signaling. Despite limitation in epigenome targeting of IL1R1, this method has potential application as a novel gene therapy for degenerative disc disease by attenuating the deleterious effect of inflammatory cytokines.

Various techniques used in gene therapy can modulate RNA and protein synthesis of the encoded genes in the recipient cells of the intervertebral disc. Autophagy, the intracellular self-digestion, and recycling system are under the negative regulation by the mammalian target of rapamycin (mTOR) gene, which can be disrupted by RNAi to downregulate catabolism in the disc (53). Although this approach focuses more on the prophylactic treatment of disc degeneration, it has the potential as a regenerative treatment over an extended period.

Contraindications

None have been listed for gene therapy as a therapeutic category. Gene therapy is usually indicated for treatment of diseases for which no cure is available, or the conventional treatments are inadequate. Some gene therapy procedures involve the use of cell implantation and administration of pharmaceutical preparations, which may have some contraindications.

Results

Worldwide clinical trials of gene therapy are listed on the NIH website: http://clinicaltrials.gov/ct2/results/map?term=gene+therapy. As of August 2021, the total number of clinical trials of gene therapy on this website was approximately 4930, including completed and ongoing trials as well as those that have been approved, but in which patient recruitment has not yet started. Except glioblastoma (101 clinical trials), other categories of neurologic disorders, with a listing of 552 clinical trials, constitute a small proportion of patients treated with gene therapy so far. These include 26 trials for Alzheimer disease, 50 for muscular dystrophy, 21 for multiple sclerosis, and 25 for Parkinson disease and parkinsonism. Several of the trials have established the safety of gene therapy, whereas phase 3 trials are in progress to establish the efficacy in some diseases such as glioblastoma. RNA interference is a sort of gene therapy and is applicable to targets in the brain that are not amenable to treatment with drugs such as small molecules, antibodies, and other proteins that have problems of delivery to the target. Vectors used in gene therapy may also be used for delivery of short interfering RNAs. Challenges of developing gene therapy for neurologic disorders include targeted delivery to the intended site of action across the blood-brain barrier, control as well as maintenance of gene expression at optimal levels, and safety of the gene products. A workshop has dealt with the following issues related to gene therapy clinical trials (43):

Adverse effects

Gene therapy has been studied extensively in animal experiments and human clinical trials. Gene therapy is generally considered to be safe now. However, the following are some of the adverse effects that have been observed.

Retroviral vectors. Although the viruses are made replication-defective, the possibility of production of a replication-competent virus remains. Stable transformation of target cells makes it is difficult to reverse the treatment if appearance of undesirable side effects requires that the treatment be stopped.

Herpes simplex virus vectors. The issue of neuronal cytotoxicity remains unsolved. Novel recombinant herpes simplex virus-1 vectors that appear to reduce the risk of neurovirulence and cytotoxicity also are less effective. Further studies on the source of herpes simplex virus-1 cytotoxicity and improved vector development are required.

Adenoviral vectors. Most of the adverse effects of viral vectors are associated with adenoviruses and some of these are:

Inflammatory effects. These have been observed mostly in the respiratory tract after local administration of adenoviral vectors.

Induction of immune response. One major concern about using adenoviral vectors for repetitive gene delivery is the induction of immune response to the vector that impedes effective gene transduction. Alternate use of adenovirus vectors from different serotypes within the same subgroup can circumvent anti-adenovirus humoral immunity to permit effective gene transfer after repeat administration although the chronicity of the expression is limited by cellular immune processes directed both against the transgene and the viral gene products expressed by the vector. Animal experimental data show the potential of immunosuppression in the management of immune reactions triggered by adenoviral vectors by use of monoclonal antibodies.

Undesirable effects of in vivo adenoviral vectors for arterial gene transfer. In patients with prior exposure to adenovirus, adenoviral vector-mediated arterial gene transfer might affect the success of arterial gene transfer and the duration of recombinant gene expression as well as the likelihood of a destructive immune response to the transduced cells.

Death due to hepatotoxicity. One death was reported in 1999 in a patient receiving adenoviral-mediated gene therapy for a hepatic metabolic disorder.

Neurotoxicity of adenoviral vectors for brain tumor therapy. Adenoviral vector-mediated herpes simplex virus-thymidine kinase gene transfer combined with ganciclovir is being considered as a therapy for malignant brain tumors. The aim is to develop a more effective and less toxic vector for brain tumor therapy. The major concern is the toxicity of adenovirus, which has not yet been resolved by animal experiments. However, a phase 1 clinical trial on patients with glioblastoma is planned to determine if there is significant toxicity. E-1 deleted adenoviral vectors trigger a strong inflammatory response in the brain but this immune response is not sufficient to eliminate complete expression of genes encoded by the adenoviral construct. Injection of adenoviral vector carrying a thymidine kinase gene produces viral meningitis (as seen on histopathological examination) when injected into the cerebrospinal fluid space in experimental animals including nonhuman primates.

Toxicity of lipopolysaccharides. The presence of lipopolysaccharides as a contaminant in plasmid prepared from Escherichia coli is well documented. Lipopolysaccharides are internalized during adenovirus-mediated gene transfer and can generate toxicity in primary cell types. Other commonly used nonviral methods of gene transfer can also generate toxicity in some primary cell types. Toxicity has been found to be due to lipid A component of lipopolysaccharides. The lipopolysaccharides-chelating antibiotic polymyxin B can block this activity.

Management of neuroinflammatory responses to AAV-mediated gene therapies. Neuroinflammatory responses after CNS-targeted delivery of large doses of AAV-mediated gene therapies are a critical concern. Strategies to reduce immune responses at various stages during gene therapy include the following (46):

Measures to improve safety and efficacy of gene therapy. Ability to direct gene transfer vectors to specific target cells is an important task to be tackled and will be important not only to achieve a therapeutic effect but also to limit potential adverse effects. Considerable efforts are being made to develop nonviral vectors to avoid some of the adverse effects of viral vectors.

Special considerations

Pregnancy. Gene therapy is relevant to pregnancy because fetal gene therapy is feasible. In the case of neurogenetic disorders, therapeutic efforts should be aimed at interventions in fetal life to prevent early disease manifestations. Prenatal diagnosis has improved and surgical interventions on the fetus are already established. Fetal gene therapy offers the following advantages:

Ethical aspects of gene therapy. A detailed discussion of ethics is beyond the scope of this article. In practice, ethical aspects of public attitudes are worth considering. There has been an increasing acceptance of gene therapy in North America during the past quarter of a century since first gene therapy trials on humans. Results of an opinion survey show that most of the respondents (greater than 90%) accept gene therapy as a treatment for severe illnesses such as Alzheimer disease, but this receptivity decreases for conditions perceived as less severe such as attention deficit hyperactivity disorder (79%), and for nontherapeutic applications (47%) such as enhancement of performance in healthy persons (49). The greatest area of concern for the application of gene therapy in neurologic disorders is the fear of not receiving sufficient information before undergoing the treatment.

Future of gene therapy for neurologic disorders. Gene therapies are in development for neurodegenerative disorders, neurogenetic disorders, and neuromuscular disorders, and many of these are in clinical trials with potential for therapeutic applications. Some therapies have already been approved for clinical use. The scope of gene therapy extends beyond genetic disorders. Genetically engineered stem cells could be used to repair tissue function for regeneration in neurologic disorders.

Few medical centers and hospitals have sufficiently experienced teams to participate in gene therapy clinical trials for neurologic conditions because gene therapy is not part of training programs for most neurosurgeons and neurologists. Expanded indications and demand for CNS gene therapies will require a worldwide educational effort to supplement the training of clinical neuroscientists, and a few centers of excellence will need to establish relevant educational training requirements and best practice for such therapeutic approaches (20).

Gene delivery is the biggest hurdle for effective gene therapy. Both intrathecal and direct intracranial delivery are invasive and associated with greater risk than peripheral delivery. Therefore, efforts to improve strategies for delivery of gene therapy for CNS disorders are ongoing. Systemic and intranasal delivery methods using nanoparticle and exosome carriers are in development. Although considerable progress has been made to improve vector design, gene selection, and targeted delivery, there are some limitations such as immunogenic reactions, nonspecificity of viral vectors, and lack of validated biomarkers to track the efficacy of therapy (32). Nonviral gene transfer using closed-end linear duplex molecules have the potential to transduce cells without triggering an immune response, but it would require direct injection into the brain.

In terms of specific vectors for gene therapy, adeno-associated viruses are favored as they do not integrate into the host genome appreciably and are derived from a nonpathogenic virus. The first gene therapy approved for Leber amaurosis is driven by a specific genetic defect. It is in a confined location (ie, retina) and is easier to treat with adeno-associated virus-mediated gene therapy than more widely distributed pathologies of neurodegenerative disorders. Other gene therapy products are expected to be approved in the next few years.

Antisense gene therapy has already become a viable treatment option for neurologic disease. Nusinersen, a splice altering antisense oligonucleotide, is approved for the treatment of spinal muscular atrophy. Other antisense oligonucleotides are in development for Huntington disease and amyotrophic lateral sclerosis.

Gene editing technologies, particularly using CRISPR-Cas, will play an important role in the future of gene therapy for neurologic disorders. Successful genome editing has been demonstrated across a range of different brain cells including neurons, astrocytes, and microglia through fluorescence imaging.

Highly penetrant genes associated with neurodevelopmental disorders are targets for gene therapy, but development of techniques for these is challenging because they are highly polygenic (42).

Scientific basis

Gene therapy involves the introduction of genetic material into cells to replace or supplement defective genes, or to induce the expression of a novel gene to alleviate a disease. Both viral and nonviral vectors have been used for this purpose. Viral vectors for gene delivery to the nervous system include adeno-associated virus, retrovirus, adenovirus, lentivirus, and herpes virus. In addition to the use of an appropriate vector, the choice of a proper transgene to treat a neurologic disorder is also critical. Properties of different viruses offer unique solutions to the challenges of gene therapy, such as cell targeting, transgene expression, and vector production, and it is important to consider these when selecting the most effective vector for a specific therapeutic function (40).