**1. Introduction**

#### **1.1. Concept and historical evolution of gene therapy**

Gene therapy can be broadly defined as the introduction of genetic material into target cells in order to modify and control protein expression for therapeutic or experimental purposes [1]. Nowadays, the culmination of the Human Genome Project along with recent advances in molecular biology have provided a better understanding of cellular and pathogenic processes, and several genes have been identified as targets for therapeutic approaches. Additionally, the constant advance in the development of gene carriers for the delivery of nucleic acids into target cells has led to conceiving new therapeutic strategies for the treatment of pathologies by genetic and cell-based approaches, collectively known as gene therapy [1].

Researchers have been working for decades to bring gene therapy to the clinic, but very few patients have received an effective gene-therapy treatment. The potential of gene therapy in medical applications was recognized soon after the discovery of DNA as genetic material, and the concept of gene therapy arose during the 1960s and 1970s [2]. The first success of gene therapy on humans arrived in 1990, it was performed by researchers at the National Institute of Health, and the treated disease was a form of severe combined immune deficiency (SCID) due to defects in the gene encoding adenosine deaminase (ADA) [3]. However, a fatal event in 1991 raised serious concerns about gene therapy. An eighteen-year-old boy died as a result of his voluntary participation in a gene therapy trial, becoming the first known human victim of this technology [4]. The Food and Drug Administration (FDA) investigation concluded that the scientists involved in the trial did not foresee serious side effects or fatality and that they did not follow the federal rules to ensure the safety of the participants [4]. This tragic case caused a severe setback in the research field of gene therapy.

According to data updated to June 2014 and presented by *The Journal of Gene Medicine* [5], since the onset of the first gene therapy clinical trial in 1989, more than 2000 new clinical trials for gene therapy have been approved globally (Figure 1). As shown in Figure 2, these trials address the most challenging diseases of today, that is, cancer (64.1% of approved trials), monogenic diseases (9.1%) such as cystic fibrosis, infectious diseases (8.2%) and cardiovascular diseases (7.8%). Although in a lesser extent, neurological diseases (1.8%) and ocular diseases (1.6%) are also subject to clinical trials with gene therapy. However, despite the intensive study during the last few years, at present only 0.1% of all the gene therapy products approved for clinical trials have arrived to the phase IV (Figure 3). In 2012, the European Medicine Agency approved for the first time a gene therapy product, Glybera, an adeno-associated viral vector engineered to express lipoprotein lipase in the muscle for the treatment of lipoprotein lipase deficiency [5].

#### **1.2. The need of carriers**

One of the main reasons why gene therapy clinical trials are still few in number is the lack of suitable and safe approaches to deliver the genetic material to target cells. The success of gene therapy critically depends on suitable transfection vectors, which should be able to: (i) protect nucleic acids against degradation by blood and interstitial nucleases, (ii) promote internaliza‐ tion of the genetic material into target cells and (iii) release the nucleic acids once inside the cell to the correct site [1]. Furthermore, an ideal gene delivery system should be effective, specific, long-lasting, safe, easy to use and as inexpensive as possible [6]. Broadly, gene delivery vectors are mainly classified into two categories: viral vectors and non-viral vectors. According to data updated to June 2014 and presented by *The Journal of Gene Medicine* [7], among the over 2,000 clinical trials for gene therapy approved globally nowadays, 70% correspond to trials using viral vectors. As shown in Figure 4, there is a 17.7% of the gene therapy clinical trials that use naked DNA, and 5.3% of trials use lipofection [7].

**1. Introduction**

146 Gene Therapy - Principles and Challenges

**1.1. Concept and historical evolution of gene therapy**

Gene therapy can be broadly defined as the introduction of genetic material into target cells in order to modify and control protein expression for therapeutic or experimental purposes [1]. Nowadays, the culmination of the Human Genome Project along with recent advances in molecular biology have provided a better understanding of cellular and pathogenic processes, and several genes have been identified as targets for therapeutic approaches. Additionally, the constant advance in the development of gene carriers for the delivery of nucleic acids into target cells has led to conceiving new therapeutic strategies for the treatment of pathologies

Researchers have been working for decades to bring gene therapy to the clinic, but very few patients have received an effective gene-therapy treatment. The potential of gene therapy in medical applications was recognized soon after the discovery of DNA as genetic material, and the concept of gene therapy arose during the 1960s and 1970s [2]. The first success of gene therapy on humans arrived in 1990, it was performed by researchers at the National Institute of Health, and the treated disease was a form of severe combined immune deficiency (SCID) due to defects in the gene encoding adenosine deaminase (ADA) [3]. However, a fatal event in 1991 raised serious concerns about gene therapy. An eighteen-year-old boy died as a result of his voluntary participation in a gene therapy trial, becoming the first known human victim of this technology [4]. The Food and Drug Administration (FDA) investigation concluded that the scientists involved in the trial did not foresee serious side effects or fatality and that they did not follow the federal rules to ensure the safety of the participants [4]. This tragic case

According to data updated to June 2014 and presented by *The Journal of Gene Medicine* [5], since the onset of the first gene therapy clinical trial in 1989, more than 2000 new clinical trials for gene therapy have been approved globally (Figure 1). As shown in Figure 2, these trials address the most challenging diseases of today, that is, cancer (64.1% of approved trials), monogenic diseases (9.1%) such as cystic fibrosis, infectious diseases (8.2%) and cardiovascular diseases (7.8%). Although in a lesser extent, neurological diseases (1.8%) and ocular diseases (1.6%) are also subject to clinical trials with gene therapy. However, despite the intensive study during the last few years, at present only 0.1% of all the gene therapy products approved for clinical trials have arrived to the phase IV (Figure 3). In 2012, the European Medicine Agency approved for the first time a gene therapy product, Glybera, an adeno-associated viral vector engineered to express lipoprotein lipase in the muscle for

One of the main reasons why gene therapy clinical trials are still few in number is the lack of suitable and safe approaches to deliver the genetic material to target cells. The success of gene

by genetic and cell-based approaches, collectively known as gene therapy [1].

caused a severe setback in the research field of gene therapy.

the treatment of lipoprotein lipase deficiency [5].

**1.2. The need of carriers**

**Figure 1.** Number of gene therapy clinical trials approved worldwide 1989-2014 (adapted from http:// www.wiley.co.uk/genmed/clinical).

Despite the still absolute predominance of viral-vector-based gene delivery platforms – which is due to their higher transfection efficiency – in clinical trials, non-viral vectors represent promising and safer alternatives to viruses. In addition, non-invasive routes of administration for gene delivery systems are currently being studied, such as intranasal administration to target the brain, topical administration on the surface of the eye to treat retinal inherited diseases and aerosolized formulations for inhalation for the treatment of pulmonary diseases. There is reasonable hope to suggest that future gene delivery systems might be based on effective non-viral vectors administered through non-invasive routes and that they would constitute a safe, easy to produce, cheap and customizable alternative to gene delivery platforms. Moreover, it is increasingly accepted that future gene delivery platforms may be based on multifunctional vectors specifically tailored for different applications [1].

**Figure 2.** Indications addressed by gene therapy clinical trials (adapted from http://www.wiley.co.uk/genmed/clinical).

**Figure 3.** Phases of gene therapy clinical trials (adapted from http://www.wiley.co.uk/genmed/clinical).

**Figure 4.** Vectors used in gene therapy in clinical trials (adapted from http://www.wiley.co.uk/genmed/clinical).

#### *1.2.1. Viral vectors*

**Indications addressed by gene therapy clinical trials**

148 Gene Therapy - Principles and Challenges

**Figure 2.** Indications addressed by gene therapy clinical trials (adapted from http://www.wiley.co.uk/genmed/clinical).

**Phases of gene therapy clinical trials**

**Figure 3.** Phases of gene therapy clinical trials (adapted from http://www.wiley.co.uk/genmed/clinical).

Cancer diseases 64.1%

Monogenic diseases 9.1%

Infectious diseases 8.2%

Cardiovascular diseases 7.8%

Neurological diseases 1.8%

Ocular diseases 1.6% (n=33)

Inflammatory diseases 0.7%

Phase I 59.2% (n=1230) Phase I/II 19.3% (n=400) Phase II 16.7% (n=346) Phase II/III 1% (n=20) Phase III 3,6% (n=75) Phase IV 0,1% (n=2) Single subject 0,1% (n=3)

Others 6,8 % (n=141)

(n=1331)

(n=188)

(n=170)

(n=162)

(n=37)

(n=14)

In this review we will focus on non-viral-vector-based strategies for gene delivery platforms, but we will briefly discuss the most relevant aspects of viral gene therapy. Viruses are highly evolved biological machines that efficiently gain access into host cells, deliver their genetic material to cells and exploit the cellular machinery to facilitate their replication [8]. Therefore, viruses represent an excellent platform for the development of recombinant vectors containing foreign genes for gene delivery purposes [1]. However, viral vectors also present many impediments such as the low carrying capacity, the expensive and complex production and, most importantly, safety issues, since they can induce oncogenesis when randomly integrated in the host genome. In addition, the human immune system recognizes and combats viruses, shortening their effectiveness [1]. Table 1 summarizes the principal viral vectors used in gene therapy, as well as their main utilities and impediments. Viral vectors are, therefore, powerful tools but present important drawbacks for clinical use in humans.

#### *1.2.2. Non-viral vectors*

Non-viral vectors have emerged as a safer, cheaper and easier-to-produce alternative to viral vectors. In fact, non-viral vectors can be produced on a large scale with high reproducibility and acceptable costs, they are relatively stable to storage, they can be administered repeatedly with no or little immune response and the dimension of the genetic material they can carry is practically unlimited [1,9]. Nevertheless, the employment of non-viral gene delivery vectors is still strongly limited by their lower transfection efficiency as compared to viral vectors [1].


**Table 1.** Principal viral vectors used in gene therapy, advantages and drawbacks. (Based on [8])

Non-viral vectors can be classified into two main categories depending on whether they are based on physical methods or on chemical methods. We will briefly review the most commonly employed non-viral DNA delivery systems for each category.

#### *1.2.2.1. Physical methods*

Physical methods for gene delivery purposes usually employ physical force to create transient membrane holes to cross the cell membrane and enhance gene transfer [6,10]. No particulate system is used to introduce the genetic material into the target cells [6]. Needle injection, ballistic DNA injection, electroporation, sonoporation, photoporation, magnetofection and hydroporation are the most utilized physical methods at present [6,11].

#### *1.2.2.2. Chemical methods*

Depending on the chemical feature, those methods can be classified into three groups: cationic lipids, cationic polymers and inorganic nanoparticles. Chemical vectors based on cationic lipids and cationic polymers form condensed complexes with negatively charged DNA through electrostatic interactions [10]. The complexes protect DNA and facilitate cell uptake intracellular delivery [10]. The principal characteristics of the non-viral chemical vectors are the following:

**•** *Vectors based on cationic lipids*

As shown in Figure 4, cationic lipid-mediated gene transfer or lipofection represents the most commonly used non-viral gene delivery system. Cationic lipids share four common functional domains: (i) a hydrophilic head-group, which is responsible for the interaction with the DNA; (ii) a hydrophobic domain, which is usually derived from aliphatic hydrocarbon chains; (iii) a linker structure, which influences the flexibility, stability and biodegradability of the cationic lipid; and (iv) a backbone domain, which separates the polar head-group from the hydrophobic domain and it is usually a serinol or a glycerol group [12]. Changes in those domains can vary the transfection efficiency of different vectors elaborated with cationic lipids. The most employed cationic lipid formulations for gene delivery platforms are: (i) **liposomes** – vesicles made up of phospholipids; (ii) **niosomes** –non-ionic surfactant vesicles, with greater physicchemical and storage stability than liposomes; and (iii) **solid lipid nanoparticles (SLN)** – particles with a solid lipid core, stabilized with surfactants [13].

#### **•** *Vectors based on cationic polymers*

Non-viral vectors can be classified into two main categories depending on whether they are based on physical methods or on chemical methods. We will briefly review the most commonly

**Episomal Utility Impediments**

in most tissues

expression



inflammatory response






response


response

cells

Physical methods for gene delivery purposes usually employ physical force to create transient membrane holes to cross the cell membrane and enhance gene transfer [6,10]. No particulate system is used to introduce the genetic material into the target cells [6]. Needle injection, ballistic DNA injection, electroporation, sonoporation, photoporation, magnetofection and

Depending on the chemical feature, those methods can be classified into three groups: cationic lipids, cationic polymers and inorganic nanoparticles. Chemical vectors based on cationic lipids and cationic polymers form condensed complexes with negatively charged DNA through electrostatic interactions [10]. The complexes protect DNA and facilitate cell uptake intracellular delivery [10]. The principal characteristics of the non-viral chemical vectors are

As shown in Figure 4, cationic lipid-mediated gene transfer or lipofection represents the most commonly used non-viral gene delivery system. Cationic lipids share four common functional

employed non-viral DNA delivery systems for each category.

**Table 1.** Principal viral vectors used in gene therapy, advantages and drawbacks. (Based on [8])

**Adenovirus** Episomal - Very efficient transfection

**(AAV)** Episomal (>90%) - Not inducing

**Retrovirus** Integrative - Persistent gene

hydroporation are the most utilized physical methods at present [6,11].

*1.2.2.1. Physical methods*

**Viral vector Integrative/**

150 Gene Therapy - Principles and Challenges

**Lentivirus** Integrative

**Virus-1 (HSV-1)** Episomal

**Adeno-associated virus**

**Herpes Simplex**

*1.2.2.2. Chemical methods*

**•** *Vectors based on cationic lipids*

the following:

Vectors based on cationic polymers are mostly spherical particles ranging in the size 1-1000 nm and they condense DNA into polyplexes preventing DNA from degradation [6]. The DNA can be entrapped in the polymeric matrix or can be adsorbed or conjugated on the surface of the nanoparticles [6]. The most popular cationic polymers employed for DNA delivery purposes are: (i) poly(ethylene imine) – PEI, which has an excellent buffering capacity; (ii) chitosan – a linear polysaccharide derived from the deacetylation of the natural chitin; (iii) cyclodextrins – a series of natural cyclic oligosaccharids; (iv) dendrimers – tree-shaped synthetic molecules up to a few nanometers in diameter that are formed with a regular branching structure; and (vi) Poly(L-lysine) – PLL, which can form nanometer-size complexes with polynucleotides thanks to the presence of protonable amine groups on the lysine moiety [6,13,14].

#### **•** *Vectors based on inorganic nanoparticles*

Inorganic nanoparticles are nanostructures varying in size, shape and porosity, and calcium phosphate, silica, gold, and several magnetic compounds are the most studied [6,15]. Inorganic particles can be easily prepared and surface-functionalized. They exhibit good storage stability and are not subject to microbial attack [6,16].

In summary, non-viral vectors for gene delivery represent a safer alternative to conventional viral vectors. However, although tremendous progress has been made in this field in recent years, the clinical application of non-viral-vector-based gene therapy is still hampered by the lack of effective gene delivery techniques. In the present review, we will discuss the up-to-date and possible future strategies to improve DNA transfer efficacy using non-viral vectors and focusing on non-invasive routes of administration. First, the intracellular barriers that nonviral vectors have to overcome and the strategies to improve the transfection efficiency in this regard will be described. Second, we will review the extracellular barriers that hamper an efficient gene delivery, as well as the invasive and the alternative non-invasive routes of administration that elude those barriers. Finally, challenges for non-viral vectors to reach clinical trials will be discussed, focusing on the transfection efficiency, the targeting and the duration of the transfected gene expression.
