**4. Challenges of non-viral gene therapy and future prospects**

Non-viral gene therapy has emerged as a promising therapeutic approach for gene delivery. Even if this field is still far from clinical practice, much progress has been made in the last few years regarding both the optimization of the non-viral vector formulations and the exploration of alternative routes of administration. For transgene expression to occur, optimal non-viral vectors should not elicit an immune response and should be able, among other aspects, to protect the DNA cargo from degradation in circulation, to enable extravasation from the bloodstream, to traverse cellular membranes, to enhance endosomal escape and to facilitate DNA transport to the nucleus [84]. The comprehensive understanding of the extracellular and intracellular barriers that vector/DNA complexes have to overcome in order to achieve an efficient transfection, has allowed the development of several strategies to surpass all those barriers, most of them based on formulation modifications of the complexes and on the use of local routes of administration. Regarding this latter aspect, considerable evidence suggests that the optimization of non-invasive routes of administration may provide safer and more effective gene delivery platforms in the future; therefore, it might be relevant to guide some efforts in this direction.

The major limitation of non-viral gene delivery, as mentioned repeatedly, is the low transfec‐ tion efficiency. Several strategies discussed along the chapter increase transfection efficiency by providing the nanoparticles the ability to overcome extra- and intracellular barriers. However,therearetwootheraspects thatarealsoessentialfordevelopingoptimalgenedelivery platforms: *targeting* and *long-term expression* of the transgene. Both are crucial to bring nonviral gene delivery systems into the clinic, since they provide specificity and sustained effect of the treatment, respectively. Many efforts have been made in this regard; however, further research is still required. In addition, other aspects such as the toxicity of the nanoparticles and the manufacturing and regulatory issues have to be carefully considered. Here, we will briefly discuss the current strategies for targeting the desired cells or tissues and for achieving a longterm expression of the transgene. We will also highlight the importance of considering the toxicity, manufacturing and regulatory issues of the nanoparticle formulations.

#### **4.1. Targeting**

Targeting to the desired cells or tissue can be achieved by modifying either the vehicle (the non-viral vector) or the cargo (the plasmid DNA). The most employed strategy is the attach‐ ment to the non-viral vector specific ligands (such as transferrin for targeting the SNC) that recognize particular receptors present in the target cells or tissues. As discussed earlier, this approach has proved effective in several studies. Also, in cancer gene therapy, some strategies are based on the exploitation of the tumour-specific physiological changes (the tumour microenvironment) to specifically conduct the nanoparticles to cancer cells [85].

On the other hand, another possibility is to introduce modifications in the DNA cargo (instead of the vector) to achieve targeted expression of the transgene, this approach is known as "transcriptional targeting." This strategy is based on the use of DNA expression cassettes that contain regulatory regions that are recognized by transcription factors specifically present or selectively expressed by the target cell population [85]. In this strategy the DNA would, in theory, be delivered to all tissues, but the expression of the transgene would only occur in the cell populations where the particular transcription factors are present, that is, in the target cell populations [84]. The success of this method of targeting needs prior knowledge of a difference in transcription factor expression between the target and normal tissue [84].

Targeting is an essential requirement in gene delivery systems. Beneficial aspects of targeted gene delivery include, among others, increased bioavailability ofthe therapeutic productin the diseased tissue; reduced accumulation in healthy tissues and, hence, reduced side effects; reduction of drug dosage and reduced dosing frequency, which enhances patient compli‐ ance. All these aspects help to increase the therapeutic efficacy and permit to reduce treat‐ ment costs [86].

#### **4.2. Duration of gene expression**

bloodstream, to traverse cellular membranes, to enhance endosomal escape and to facilitate DNA transport to the nucleus [84]. The comprehensive understanding of the extracellular and intracellular barriers that vector/DNA complexes have to overcome in order to achieve an efficient transfection, has allowed the development of several strategies to surpass all those barriers, most of them based on formulation modifications of the complexes and on the use of local routes of administration. Regarding this latter aspect, considerable evidence suggests that the optimization of non-invasive routes of administration may provide safer and more effective gene delivery platforms in the future; therefore, it might be relevant to guide some

The major limitation of non-viral gene delivery, as mentioned repeatedly, is the low transfec‐ tion efficiency. Several strategies discussed along the chapter increase transfection efficiency by providing the nanoparticles the ability to overcome extra- and intracellular barriers. However,therearetwootheraspects thatarealsoessentialfordevelopingoptimalgenedelivery platforms: *targeting* and *long-term expression* of the transgene. Both are crucial to bring nonviral gene delivery systems into the clinic, since they provide specificity and sustained effect of the treatment, respectively. Many efforts have been made in this regard; however, further research is still required. In addition, other aspects such as the toxicity of the nanoparticles and the manufacturing and regulatory issues have to be carefully considered. Here, we will briefly discuss the current strategies for targeting the desired cells or tissues and for achieving a longterm expression of the transgene. We will also highlight the importance of considering the

Targeting to the desired cells or tissue can be achieved by modifying either the vehicle (the non-viral vector) or the cargo (the plasmid DNA). The most employed strategy is the attach‐ ment to the non-viral vector specific ligands (such as transferrin for targeting the SNC) that recognize particular receptors present in the target cells or tissues. As discussed earlier, this approach has proved effective in several studies. Also, in cancer gene therapy, some strategies are based on the exploitation of the tumour-specific physiological changes (the tumour

On the other hand, another possibility is to introduce modifications in the DNA cargo (instead of the vector) to achieve targeted expression of the transgene, this approach is known as "transcriptional targeting." This strategy is based on the use of DNA expression cassettes that contain regulatory regions that are recognized by transcription factors specifically present or selectively expressed by the target cell population [85]. In this strategy the DNA would, in theory, be delivered to all tissues, but the expression of the transgene would only occur in the cell populations where the particular transcription factors are present, that is, in the target cell populations [84]. The success of this method of targeting needs prior knowledge of a difference

Targeting is an essential requirement in gene delivery systems. Beneficial aspects of targeted gene delivery include, among others, increased bioavailability ofthe therapeutic productin the diseased tissue; reduced accumulation in healthy tissues and, hence, reduced side effects;

toxicity, manufacturing and regulatory issues of the nanoparticle formulations.

microenvironment) to specifically conduct the nanoparticles to cancer cells [85].

in transcription factor expression between the target and normal tissue [84].

efforts in this direction.

168 Gene Therapy - Principles and Challenges

**4.1. Targeting**

Long-term or sustained expression of the transgene delivery constitutes a real challenge in non-viral gene therapy, and it is a considerable limiting factor, since transient expression requires repeated dosing and makes the therapeutic effect unsustained. Transgene expression can decrease in time due to several factors, including destruction by nucleases, loss by recombination, distribution to non-nuclear compartments and/or recognition and subsequent silencing of foreign DNA [85]. In addition, in dividing cells the percentage of transfected cells decreases at each division, because while cells replicate, plasmids do not.

Strategies to increase duration of transgene expression have focused on plasmid DNA modifications rather than on vector modifications. Some of those strategies are aimed at integrating the transgenes into the host genome using viral integrases, site-specific recombi‐ nases and transposases, which are enzymes with capacity of inserting foreign DNA into the host genome [85]. However, this approach cannot be clinically applicable in humans because of its associated risks, such as the induction of insertional mutagenesis in the host cells.

A different strategy to achieve sustained transgene expression is the use of autonomously replicating plasmids or episomes, which does not require integration in the host genome and, hence, avoids insertional mutagenesis risks [85]. In addition, episomally replicating plasmids usually yield high levels of transgene expression. These strategies incorporate genes that encode necessary cofactors for transcription of the plasmid to the therapeutic plasmid DNA, making the transgene expression less dependent on host factors. Incorporation of viral DNA that allows the plasmid to replicate extrachromosomally is an efficient approach, but it presents a major drawback, since those replication-inducing viral DNA elements are associated with induction of immune response and risk of transformation and oncogenicity [85]. Alternatively, mammalian scaffold/matrix attachment regions (S/MARs) have been identified that can be incorporated to plasmid DNA instead of the aforementioned viral sequences. These sequences can also enhance episomal replication of the plasmids, probably by bringing the plasmids into contact with the host replication machinery [73]. Episomally replicating plasmids are espe‐ cially important in cancer gene therapy, where maintenance and vertical transfer of the therapeutic plasmid will be essential because of the presence of dividing tumour cells [85].

Some other strategies for achieving sustained expression focus on the prevention of transgene silencing, since cellular gene silencing mechanisms can impede transgene expression [85].

Further modifications can be also applied to the therapeutic plasmid DNA in order to increase the strength or the specificity of the therapeutic transgene expression. For instance, positive feedback loops can be incorporated. To do this, a promoter that drives the expression of both the transgene and of a strong artificial transcriptional activator is used. This transcriptional activator is capable of interacting with appropriate binding sites within the promoter and, that way, upregulating transgene expression, as well as its own expression [85]. Technologies incorporating positive feedback loops are estimated to increase strength of weak but highly specific regulatory elements [85].

#### **4.3. Toxicity, manufacturing and regulatory issues**

Besides increasing transfection efficiency through targeting and other strategies, careful consideration of toxicity, manufacturing and regulatory issues of non-viral delivery systems is mandatory. Scalability and long-term storage requirements are essential factors to be taken into serious consideration when developing non-viral formulations for potential commercial application and introduction in clinical practice [19]. A generally accepted advantage of nonviral vectors is their ease of large-scale manufacture. However, this can become more complex as formulations increase in complexity, incorporating stabilizing components and bioactive targeting ligands.

Regarding toxicity of non-viral vectors, characteristics such as size, charge, surface function‐ alization, shape, and architecture may contribute to the toxicity profile of nanoparticles. Nonviral vectors are thought to cause toxicity through different mechanisms, including membrane destabilization and lysis, inducing oxidative stress, initiating inflammatory response, inducing global changes in gene expression profiles, among others [85]. Also, the properties of the biomaterials used can influence toxicity, depending on the rate of degradation and persistence in organs [19]. Persisting and accumulating biomaterials are more likely to induce an inflam‐ matory response; also, products of the degradation of nanoparticles could potentially cause toxicity. However, knowledge about how non-viral vectors are disassembled and metabol‐ ically processed is still very scarce and further research is necessary in this regard [19].

To conclude, the concept of a unique universal non-viral vector is nowadays abandoned, and it is increasingly accepted that future non-viral gene delivery platforms will be based on multifunctional vectors specifically tailored for different applications [1]. However, there are some generally assumed features that all non-viral vectors should accomplish for efficient gene delivery. In short, three main factors should be taken into consideration when developing nonviral gene delivery platforms: (i) formulation components (of both the vector and the DNA) – the nanoparticle should be able to protect DNA over extra- and intracellular barriers and deliver the cargo into the nucleus of target cells; once inside the nucleus, the plasmids can be addressed to the nuclear matrix for episomal replication and sustained expression; (ii) manufacturing issues – non-viral vector formulations should have potential for scale-up; and (iii) safety and regulatory issues – formulations should be non-toxic, non-immunogenic and should have suitable storage conditions. Although non-viral vectors are still far from clinical practice, they represent a safer alternative to conventional viral vectors. Several formulations and strategies are under investigation with the aim of overcoming extra- and intracellular barriers, enhancing targeted transfection and, in general, increasing transfection efficiency. In addition, those formulations would ideally be suitable for administration through noninvasive routes, such as the intranasal administration to target the brain, topical ocular administration for the retina and aerosols for pulmonary diseases. Finally, the inclusion of novel functional modules within both the carrier and the DNA molecule will produce a range of non-viral vectors tailored for specific applications, including the safe and long-term expression of therapeutic genes in humans [40]. Therefore, reasonable hope suggests that nextgeneration gene delivery systems may be based on non-viral vector systems tailored for specific applications and suitable for non-invasive administration routes, representing an ideal platform to effectively shuttle the genetic material to target cells in a safe and controlled way.
