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[20] Griesenbach U, Boyton RJ, Somerton L, et al. Effect of tolerance induction to immu‐ nodominant T-cell epitopes of Sendai virus on gene expression following repeat ad‐

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progenitor cell depletion. Am. J. Respir. Cell Mol. Biol., 24, 671–681

CFTR expression in the human bronchus. Nat. Genet., 2, 240–248

epithelium. Proc. Natl Acad. Sci. USA, 106, 12771–12775

tracheal epithelial cell cultures. Exp. Cell Res., 198, 69–77

treatment of cystic fibrosis. Mol Ther 2007;15:229–41

ministration. Gene Ther 2006;13:449–56

Gene Ther 2004;11:S67–75

CFTR. Science, 266, 1705–1708

Mol. Ther., 17, 285–293

101, 11S–16S

ol., 24, 662–670

2005;79:1113–24

#### **Author details**

Dimosthenis Lykouras1 , Kiriakos Karkoulias1 , Christos Tourmousoglou2 , Efstratios Koletsis2 , Kostas Spiropoulos1 and Dimitrios Dougenis2

1 Department of Pulmonary Medicine, University Hospital of Patras, Greece

2 Department of Cardiothoracic Surgery, University Hospital of Patras, Greece

#### **References**

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**6. Conclusion**

622 Gene Therapy - Tools and Potential Applications

the future.

**Author details**

Dimosthenis Lykouras1

Kostas Spiropoulos1

**References**

Gene therapy is a very promising tool for the respiratory clinician and a few clinical trials have been performed. All these trials have shown safety but intermittent efficacy. Gene ther‐ apy for pulmonary diseases has not yet reached the point of clinical practice. But we can say that this tool will find a very interesting role in our efforts for treating respiratory diseases in

, Christos Tourmousoglou2

, Efstratios Koletsis2

,

, Kiriakos Karkoulias1

1 Department of Pulmonary Medicine, University Hospital of Patras, Greece

2 Department of Cardiothoracic Surgery, University Hospital of Patras, Greece

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2005;23(16S): 2586

**Chapter 26**

**Gene Therapy in Critical Care Medicine**

Critical care medicine is directed toward patients with a wide spectrum of illnesses. These have the common denominators of marked exacerbation of an existing disease, severe acute new problems, or severe complications from disease or treatments. In recent years has been an explosion of evidence based medicine with improvement in outcome, however there are several conditions in critical care patients that maintains a high morbidity and high mortali‐ ty that is necessary to be addressed [1]. Of these, severe sepsis and the acute respiratory dis‐ tress syndrome (ARDS), including acute lung injury (ALI) (syndromes consisting of acute respiratory failure associated with pulmonary infiltrates due to intra- or extra-pulmonary diseases) are two important conditions that have increased mortality in critical care units

In 1991, a Consensus Conference of the American College of Chest Physicians an the Society for Critical Care (ACCP-SCCM) introduced the term systemic inflammatory response syn‐ drome (SIRS) as the presence of at least two of four clinical criteria: body temperature more than 38˚C or less than 36˚C, heart rate more than 90 beats per minute, respiratory rate more than 20 breaths per minute or hyperventilation with PaCO<sup>2</sup> less than 32 mmHg, white blood

trophils [4]. In 2001, a new consensus suggests that other signs and symptoms could reflect the clinical response to infection, including: fever/hypothermia, tachypnea/respiratory alka‐ losis, positive fluid balance/edema, general inflammatory reaction, altered white blood count, increased biomarkers (C-reactive protein, IL-6, pro-calcitonin), hemodynamic altera‐ tions, arterial hypotension, tachycardia, increased cardiac outflow/low systemic vascular re‐ sistance/high venous saturation O2, altered skin perfusion, decreased urine output,

> © 2013 Moreno-González and Zarain-Herzberg; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

or with more than 10% immature neu‐

, less than 4000/mm<sup>3</sup>

Gabriel J. Moreno-González and

Additional information is available at the end of the chapter

Angel Zarain-Herzberg

http://dx.doi.org/10.5772/52701

**1. Introduction**

around the world [2, 3].

cell count more than 12000/mm<sup>3</sup>

properly cited.

### **Gene Therapy in Critical Care Medicine**

Gabriel J. Moreno-González and Angel Zarain-Herzberg

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52701

#### **1. Introduction**

Critical care medicine is directed toward patients with a wide spectrum of illnesses. These have the common denominators of marked exacerbation of an existing disease, severe acute new problems, or severe complications from disease or treatments. In recent years has been an explosion of evidence based medicine with improvement in outcome, however there are several conditions in critical care patients that maintains a high morbidity and high mortali‐ ty that is necessary to be addressed [1]. Of these, severe sepsis and the acute respiratory dis‐ tress syndrome (ARDS), including acute lung injury (ALI) (syndromes consisting of acute respiratory failure associated with pulmonary infiltrates due to intra- or extra-pulmonary diseases) are two important conditions that have increased mortality in critical care units around the world [2, 3].

In 1991, a Consensus Conference of the American College of Chest Physicians an the Society for Critical Care (ACCP-SCCM) introduced the term systemic inflammatory response syn‐ drome (SIRS) as the presence of at least two of four clinical criteria: body temperature more than 38˚C or less than 36˚C, heart rate more than 90 beats per minute, respiratory rate more than 20 breaths per minute or hyperventilation with PaCO<sup>2</sup> less than 32 mmHg, white blood cell count more than 12000/mm<sup>3</sup> , less than 4000/mm<sup>3</sup> or with more than 10% immature neu‐ trophils [4]. In 2001, a new consensus suggests that other signs and symptoms could reflect the clinical response to infection, including: fever/hypothermia, tachypnea/respiratory alka‐ losis, positive fluid balance/edema, general inflammatory reaction, altered white blood count, increased biomarkers (C-reactive protein, IL-6, pro-calcitonin), hemodynamic altera‐ tions, arterial hypotension, tachycardia, increased cardiac outflow/low systemic vascular re‐ sistance/high venous saturation O2, altered skin perfusion, decreased urine output,

<sup>© 2013</sup> Moreno-González and Zarain-Herzberg; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

hyperlactacemia, signs of organ dysfunction, hypoxemia, coagulation abnormalities, altered mental status, hyperglycemia, thrombocytopenia, disseminated intravascular coagulation, altered liver function, intolerance to feeding [5].

**2. Physiopathology of sepsis**

and c) RIG-I-like receptors (RLRs) [22].

initiating TLR-induced signal transduction [27].

TLR signaling cascades [28-33].

Microorganisms express macromolecular motifs, named pathogen-associated molecular pat‐ terns (PAMs) such as lipopolysaccharide (LPS), flagellin, double-stranded RNA and CpG DNA [22]. These molecules are recognized by the immune system through a family of transmembrane or intra-cytoplasmic receptors, the pattern recognition receptors (PRRs), classi‐ fied in three general families: a) Toll-like receptors (TLRs); b) NOD-like receptors (NLRs);

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 633

The TLRs are type I integral membrane glycoproteins characterized by the extracellular do‐ mains containing varying numbers of leucine-rich-repeat (LRR) motifs and a cytoplasmic signaling domain homologous to that of the interleukin 1 receptor (IL-1R), termed the Toll/ IL-1R homology (TIR) domain [23]. Based on their primary sequences, TLRs can be divides into several subfamilies, each of which recognized related PAMPs: the subfamily of TLR1, TLR2 and TLR6 recognize lipids, whereas the highly related TLR7, TLR8, TLR9 recognize nucleic acids. TLR4 recognize a very divergent collection of ligands [24]. The NLRs proteins are implicated in the recognition of bacterial components. Proteins in this family possess LRRs that mediate ligand sensing: a nucleotide binding oligomerization domain (NOD) and a domain for the initiation of signaling such as CARDs, PYRIN of baculovirus inhibitor of apoptosis repeat (BIR) domains [25]. The retinoic-acid inducible protein-I (RIG-I) is an INFinducible protein containing CARDs and a DExD/H box helicase domain and has been iden‐ tified as a cytoplasmic detector in viral infection in the TLR3 independent manner [26]. In addition to the numerous exogenous pathogen-derived ligands that activate different TLRs, endogenous TLR ligands have been identified, including hyaluronic acid, high mobility group box-1 (HMGB1) and heat shock proteins (HSPs), termed as damaged-associated mo‐ lecular patterns (DAMPs). During tissue injury or proteolysis, extracellular matrix compo‐ nents undergo cleavage, exposing moieties that can act as ligands for TLRs and therefore

The PAM/PPR interaction leads to immune cell activation with initiation of microbe-killing systems, production and secretion of pro-inflammatory cytokines and chemokines, en‐ hanced expression of co-stimulatory receptors essential for efficient T cell activation, pro‐ duction of arachinoid acid metabolites and initiation of extrinsic coagulation cascade [28-33]. The activation of the TLR signaling originated from the cytoplasmic Toll/IL-1 receptor (TIR) domain requires the association with the TIR domain-containing adaptor protein, MyD88. With ligands binding, MyD88 recruits IL-1 receptor-associates kinase-4 (IRAK-4) to TLRs through interaction of the death domains of both molecules. IRAK-1 activated by phosphor‐ ylation then associates with TRAF6, finally leading to activation of MAP kinases and NFκB. Additional modes of regulation for these pathways include TRIF-dependent induction of TRAF6 signaling by RIP1 and negative regulation of TIRAP mediated downstream signaling by ST2L, TRIAD3A and SOCS1. MyD88-independent pathways induce activation of IRF3 and expression of interferon-β. TIR-domain containing adaptors such as TIRAP, TRIF and TRAM regulate TLR-mediated signaling pathways by providing specificity for individual

Systemic inflammatory response syndrome can result from diverse etiologies, including, but not limited to infectious, trauma, pancreatitis, ischemia-reperfusion injury, and burns [6]. Sepsis is defined as the presence of infection and some of the listed signs and symptoms of SIRS, whereas severe sepsis is defined as sepsis associated with organ dysfunction and shock septic as severe sepsis with hypotension, despite adequate fluid resuscitation [7].

Over 18 million cases of severe sepsis occur each year. The number of severe sepsis cases is set to grow a rate of 1.5% per year from the annual incidence of 3 cases per 1000 of the popu‐ lation in 2001 [8, 9]. Sepsis is a major cause of mortality throughout the world, killing ap‐ proximately 1400 people every day, being as high as an additional fifty per cent as deaths are often attributed to complications from cancer or pneumonia, and not related to sepsis [10]. Death is common among sepsis patients, with around 28-50% of patients dying within the first month of diagnosis [11-13]. Sepsis impacts the lives of many people, including the patient and their families, in addition to doctors, nursing and care staff. The intense de‐ mands made on hospital staff, equipment and facilities to treat septic patients places a sig‐ nificant burden on healthcare resources, accounting for 40% of total ICU expenditure [10]. Each year the cost of treating septic patients increases and is as high as 7.6 billion euro in Europe [10] and 17.4 billion euro in the USA [8].

One common complication of SIRS and sepsis is acute lung injury/adult respiratory distress syndrome (ALI/ARDS). According to a Join North American European consensus commit‐ tee (NAECC), ARDS is defined as an inflammatory process in the lungs with acute onset of respiratory failure, new bilateral pulmonary infiltrates on frontal chest radiograph or com‐ puted tomography, absence of left ventricular failure (clinically diagnosed or a pulmonary artery occlusion pressure <18mmHg) and hypoxemia with a ratio between the partial pres‐ sure of arterial oxygen and the fraction of inspired oxygen (PaO2/FiO2 ratio) of ≤27 kPa inde‐ pendent of the level of positive end-expiratory pressure (PEEP) [14]. ALI is defined by the same criteria except that the PaO2/FiO2 ratio is between 27 kPa and 40 kPa[14-16]. Sepsis is the most common cause of ALI/ARDS and also the most common cause of death after pa‐ tients develop ALI/ARDS [17]. The incidence of ALI/ARDS is estimated to be 20 to 50 cases per 100000 person-year, with approximately 18% to 25% of cases meeting oxygenation crite‐ ria for ALI but not for ARDS [18, 19].

The reported rate of mortality from ARDS ranges from 31% to 74% depending on the char‐ acteristics of patients, with most deaths occurring as a consequence of multiple organ failure and sepsis [18, 19]. ALI has a significant lower crude hospital mortality (32%) compared with those with ARDS (57.9%) [20]. Crude estimates of the health care costs associated with ALI/ARDS may exceed 5 billion dollars per year in the United States alone [21].

#### **2. Physiopathology of sepsis**

hyperlactacemia, signs of organ dysfunction, hypoxemia, coagulation abnormalities, altered mental status, hyperglycemia, thrombocytopenia, disseminated intravascular coagulation,

Systemic inflammatory response syndrome can result from diverse etiologies, including, but not limited to infectious, trauma, pancreatitis, ischemia-reperfusion injury, and burns [6]. Sepsis is defined as the presence of infection and some of the listed signs and symptoms of SIRS, whereas severe sepsis is defined as sepsis associated with organ dysfunction and shock septic as severe sepsis with hypotension, despite adequate fluid resuscitation [7].

Over 18 million cases of severe sepsis occur each year. The number of severe sepsis cases is set to grow a rate of 1.5% per year from the annual incidence of 3 cases per 1000 of the popu‐ lation in 2001 [8, 9]. Sepsis is a major cause of mortality throughout the world, killing ap‐ proximately 1400 people every day, being as high as an additional fifty per cent as deaths are often attributed to complications from cancer or pneumonia, and not related to sepsis [10]. Death is common among sepsis patients, with around 28-50% of patients dying within the first month of diagnosis [11-13]. Sepsis impacts the lives of many people, including the patient and their families, in addition to doctors, nursing and care staff. The intense de‐ mands made on hospital staff, equipment and facilities to treat septic patients places a sig‐ nificant burden on healthcare resources, accounting for 40% of total ICU expenditure [10]. Each year the cost of treating septic patients increases and is as high as 7.6 billion euro in

One common complication of SIRS and sepsis is acute lung injury/adult respiratory distress syndrome (ALI/ARDS). According to a Join North American European consensus commit‐ tee (NAECC), ARDS is defined as an inflammatory process in the lungs with acute onset of respiratory failure, new bilateral pulmonary infiltrates on frontal chest radiograph or com‐ puted tomography, absence of left ventricular failure (clinically diagnosed or a pulmonary artery occlusion pressure <18mmHg) and hypoxemia with a ratio between the partial pres‐ sure of arterial oxygen and the fraction of inspired oxygen (PaO2/FiO2 ratio) of ≤27 kPa inde‐ pendent of the level of positive end-expiratory pressure (PEEP) [14]. ALI is defined by the same criteria except that the PaO2/FiO2 ratio is between 27 kPa and 40 kPa[14-16]. Sepsis is the most common cause of ALI/ARDS and also the most common cause of death after pa‐ tients develop ALI/ARDS [17]. The incidence of ALI/ARDS is estimated to be 20 to 50 cases per 100000 person-year, with approximately 18% to 25% of cases meeting oxygenation crite‐

The reported rate of mortality from ARDS ranges from 31% to 74% depending on the char‐ acteristics of patients, with most deaths occurring as a consequence of multiple organ failure and sepsis [18, 19]. ALI has a significant lower crude hospital mortality (32%) compared with those with ARDS (57.9%) [20]. Crude estimates of the health care costs associated with

ALI/ARDS may exceed 5 billion dollars per year in the United States alone [21].

altered liver function, intolerance to feeding [5].

632 Gene Therapy - Tools and Potential Applications

Europe [10] and 17.4 billion euro in the USA [8].

ria for ALI but not for ARDS [18, 19].

Microorganisms express macromolecular motifs, named pathogen-associated molecular pat‐ terns (PAMs) such as lipopolysaccharide (LPS), flagellin, double-stranded RNA and CpG DNA [22]. These molecules are recognized by the immune system through a family of transmembrane or intra-cytoplasmic receptors, the pattern recognition receptors (PRRs), classi‐ fied in three general families: a) Toll-like receptors (TLRs); b) NOD-like receptors (NLRs); and c) RIG-I-like receptors (RLRs) [22].

The TLRs are type I integral membrane glycoproteins characterized by the extracellular do‐ mains containing varying numbers of leucine-rich-repeat (LRR) motifs and a cytoplasmic signaling domain homologous to that of the interleukin 1 receptor (IL-1R), termed the Toll/ IL-1R homology (TIR) domain [23]. Based on their primary sequences, TLRs can be divides into several subfamilies, each of which recognized related PAMPs: the subfamily of TLR1, TLR2 and TLR6 recognize lipids, whereas the highly related TLR7, TLR8, TLR9 recognize nucleic acids. TLR4 recognize a very divergent collection of ligands [24]. The NLRs proteins are implicated in the recognition of bacterial components. Proteins in this family possess LRRs that mediate ligand sensing: a nucleotide binding oligomerization domain (NOD) and a domain for the initiation of signaling such as CARDs, PYRIN of baculovirus inhibitor of apoptosis repeat (BIR) domains [25]. The retinoic-acid inducible protein-I (RIG-I) is an INFinducible protein containing CARDs and a DExD/H box helicase domain and has been iden‐ tified as a cytoplasmic detector in viral infection in the TLR3 independent manner [26]. In addition to the numerous exogenous pathogen-derived ligands that activate different TLRs, endogenous TLR ligands have been identified, including hyaluronic acid, high mobility group box-1 (HMGB1) and heat shock proteins (HSPs), termed as damaged-associated mo‐ lecular patterns (DAMPs). During tissue injury or proteolysis, extracellular matrix compo‐ nents undergo cleavage, exposing moieties that can act as ligands for TLRs and therefore initiating TLR-induced signal transduction [27].

The PAM/PPR interaction leads to immune cell activation with initiation of microbe-killing systems, production and secretion of pro-inflammatory cytokines and chemokines, en‐ hanced expression of co-stimulatory receptors essential for efficient T cell activation, pro‐ duction of arachinoid acid metabolites and initiation of extrinsic coagulation cascade [28-33]. The activation of the TLR signaling originated from the cytoplasmic Toll/IL-1 receptor (TIR) domain requires the association with the TIR domain-containing adaptor protein, MyD88. With ligands binding, MyD88 recruits IL-1 receptor-associates kinase-4 (IRAK-4) to TLRs through interaction of the death domains of both molecules. IRAK-1 activated by phosphor‐ ylation then associates with TRAF6, finally leading to activation of MAP kinases and NFκB. Additional modes of regulation for these pathways include TRIF-dependent induction of TRAF6 signaling by RIP1 and negative regulation of TIRAP mediated downstream signaling by ST2L, TRIAD3A and SOCS1. MyD88-independent pathways induce activation of IRF3 and expression of interferon-β. TIR-domain containing adaptors such as TIRAP, TRIF and TRAM regulate TLR-mediated signaling pathways by providing specificity for individual TLR signaling cascades [28-33].

The interaction of PAMs with NRL recruits the receptor-interacting protein-2 (RIP2) kinase activating NFκB and MAPK kinases. A number of the NRL molecules have been shown to form a complex with caspase-1 and the adaptor molecule apoptosis associated speck-like pro‐ tein containing CARD (ASC) termed inflammasome. The central effector molecule of the in‐ flammasome is the cysteine protease caspase-1 that, upon activation cleaves cytosolic pro-IL-1β, pro-IL-18 and pro-IL-33 to their active forms enabling them to be secreted into the extracellular/systemic compartments [34]. The important fact is that NRLs and TLRs may syn‐ ergize. T-cell subgroups are modified in sepsis. Helper (CD4<sup>+</sup> ) T-cells can be categorized as type 1 helper (Th1) or 2 (Th2). Th1 cells generally secrete pro-inflammatory cytokines such as tumor necrosis factor-α(TNFα) and interleukin-1β (IL-1β); Th2 cells secrete anti-inflammato‐ ry cytokines such as IL-4 and IL-10, depending on the infecting organism, the burden of infec‐ tion and other factors during sepsis may also induce apoptosis of lung and intestinal cells [35]. Activated helper T cells evolve from a Th1 phenotype, producing pro-inflammatory cyto‐ kines, to a Th2 phenotype producing anti-inflammatory cytokines [35]. In addition, apoptosis of circulating and tissue lymphocytes (B cells and CD4<sup>+</sup> T cells) contributes to immunosup‐ pression [36]. The increased pro-inflammatory cytokines, activated B cells and T cells and cir‐ culating glucocorticoid levels causes apoptosis in septic patients [37]. Increased levels of TNFα and lipopolysaccharide during sepsis may also induce apoptosis [35].

Loss of epithelial cells and endothelial cell injury are involved in pathogenesis of ALI/ARDS. The former is due to the activation of Fas related apoptosis and the secretion of cytokines and chemokines by lung epithelial cells [44]. The latter is caused by the interaction of endo‐ thelial cells with neutrophils that stimulate release of vasoactive compounds, increased pul‐ monary vascular resistance with pulmonary hypertension [45], but also endothelial cells can be directly stimulated by endotoxin via TLR-1 with the release of vasoactive mediators and molecules altering lung permeability, such as TNFα, thromboxane-A2 and endothelin-1 [46].

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 635

Resolution from lung injury is an actively regulated program involving a removal of apop‐ totic neutrophils, remodeling of matrix, clearance of protein-rich alveolar fluid [47]. Recent‐ ly, has been demonstrated that CD4+ lymphocytes as well as plasmacytoid dendritic cells are

Gene therapy is defined as the introduction of nucleic acids into cells for the purpose of altering the course of a medical condition o disease [50]. In general, the advantages of gene therapy over the other treatments are the selective treatment of affected tissues, the possibility of using locally endogenous proteins in cases where its systemic application would incur in serious adverse secondary effects, and the possibility of therapeutic long term after a single application [51]. Currently, there are three categories of gene delivery methods: viral vector based, non-viral vector based and physical methods [52]. Viralbased gene delivery systems is accomplished by using replication-deficient viruses con‐ taining the gene of interest, but with the disease-causing sequences deleted from the viral genome [53] including RNA-based viral vectors [54, 55], DNA-based viral vectors such as adenoviral vectors [56], adeno-associated viruses (AAV) vectors orherpes simplex viral vector [57].The non-viral gene delivery methods use synthetic or natural compounds or physical forces to deliver a piece of DNA into a cell [58]. Two main groups of non-viral delivery methods have developed: chemical-based, including lipofection [59] and inorgan‐ ic nanoparticles that are usually prepared from metals, inorganic salts or ceramics [60]; and using physical forces such as local or rapid systemic injection [61], particle impact

Currently, there is evidence that applying therapeutic maneuvers such as early effective an‐ tibiotic administration, intensive fluid resuscitation, mechanical ventilation in selected pa‐ tients and use of C activated protein in sickest patients improve significantly the survive in these patients [66]. There are several clinical studies that are trying to validate another kind of therapies such as extra-renal depuration, levosimendan, the use of immunoglobulins, ni‐ tric oxide, statins, selenium, the use of enteral nutrition with eicosapentaenoic acid (EPA)/ψ-

active players in this process [48, 49].

**4. Vectors for gene therapy**

[62, 63], electric pulse [64] or laser irradiation [65].

**5. Gene therapy in sepsis**

#### **3. Physiopathology of acute respiratory distress syndrome**

There are two general types of ALI/ARDS, direct and indirect. Independent of the initial in‐ sult, the final result is that alveolar-capillary barrier becomes compromised. Direct ALI/ ARDS is often associated with direct mechanical, chemical or infectious stimuli, or other di‐ rect interactions capable to induce damage to lung structures [38]. Indirect pulmonary in‐ sults such as extra-pulmonary sepsis, trauma, shock, pancreatitis, brain injury or massive transfusion are the mainly causes of indirect ALI/ARDS. However, the highest incidence of indirect ALI/ARDS is seen during sepsis.

The emigration of activated PMNs and passage through the endothelium in the lungs, one of the characteristics of ALI, is regulated via adhesion molecules. Among them, L-selectin (CD62L) on PMNs appears to be involved in the initial rolling proceed on the endothelial surface, while CD11b/CD18 on PMNs mediate a tighter contact between them. CD31 of PE‐ CAM-1 is needed in the final step for the vascular diapedesis of leukocytes [38-40]. Neutro‐ phils are able to release a variety of harmful substances, such as proteolytic enzymes, reactive oxygen/nitrogen species, cytokines and chemokynes, which may be injurious to the adjacent endothelial cell and to the alveoli [39]. PMN apoptosis is a crucial injury-limiting mechanism of inflammatory resolution. Several inflammatory agents such as LPS, TNF, IL-8, IL-6, IL-1 and granulocyte colony stimulating factor (G-CSF) can delay apoptotic re‐ sponse, providing PMN with a longer life, allowing accumulating at local tissues [41]. NFκB has been reported as a modulator of apoptosis in inflammatory cells [42, 43] allowing a proinflammatory state.

Loss of epithelial cells and endothelial cell injury are involved in pathogenesis of ALI/ARDS. The former is due to the activation of Fas related apoptosis and the secretion of cytokines and chemokines by lung epithelial cells [44]. The latter is caused by the interaction of endo‐ thelial cells with neutrophils that stimulate release of vasoactive compounds, increased pul‐ monary vascular resistance with pulmonary hypertension [45], but also endothelial cells can be directly stimulated by endotoxin via TLR-1 with the release of vasoactive mediators and molecules altering lung permeability, such as TNFα, thromboxane-A2 and endothelin-1 [46].

Resolution from lung injury is an actively regulated program involving a removal of apop‐ totic neutrophils, remodeling of matrix, clearance of protein-rich alveolar fluid [47]. Recent‐ ly, has been demonstrated that CD4+ lymphocytes as well as plasmacytoid dendritic cells are active players in this process [48, 49].

#### **4. Vectors for gene therapy**

The interaction of PAMs with NRL recruits the receptor-interacting protein-2 (RIP2) kinase activating NFκB and MAPK kinases. A number of the NRL molecules have been shown to form a complex with caspase-1 and the adaptor molecule apoptosis associated speck-like pro‐ tein containing CARD (ASC) termed inflammasome. The central effector molecule of the in‐ flammasome is the cysteine protease caspase-1 that, upon activation cleaves cytosolic pro-IL-1β, pro-IL-18 and pro-IL-33 to their active forms enabling them to be secreted into the extracellular/systemic compartments [34]. The important fact is that NRLs and TLRs may syn‐

type 1 helper (Th1) or 2 (Th2). Th1 cells generally secrete pro-inflammatory cytokines such as tumor necrosis factor-α(TNFα) and interleukin-1β (IL-1β); Th2 cells secrete anti-inflammato‐ ry cytokines such as IL-4 and IL-10, depending on the infecting organism, the burden of infec‐ tion and other factors during sepsis may also induce apoptosis of lung and intestinal cells [35]. Activated helper T cells evolve from a Th1 phenotype, producing pro-inflammatory cyto‐ kines, to a Th2 phenotype producing anti-inflammatory cytokines [35]. In addition, apoptosis

pression [36]. The increased pro-inflammatory cytokines, activated B cells and T cells and cir‐ culating glucocorticoid levels causes apoptosis in septic patients [37]. Increased levels of TNF-

There are two general types of ALI/ARDS, direct and indirect. Independent of the initial in‐ sult, the final result is that alveolar-capillary barrier becomes compromised. Direct ALI/ ARDS is often associated with direct mechanical, chemical or infectious stimuli, or other di‐ rect interactions capable to induce damage to lung structures [38]. Indirect pulmonary in‐ sults such as extra-pulmonary sepsis, trauma, shock, pancreatitis, brain injury or massive transfusion are the mainly causes of indirect ALI/ARDS. However, the highest incidence of

The emigration of activated PMNs and passage through the endothelium in the lungs, one of the characteristics of ALI, is regulated via adhesion molecules. Among them, L-selectin (CD62L) on PMNs appears to be involved in the initial rolling proceed on the endothelial surface, while CD11b/CD18 on PMNs mediate a tighter contact between them. CD31 of PE‐ CAM-1 is needed in the final step for the vascular diapedesis of leukocytes [38-40]. Neutro‐ phils are able to release a variety of harmful substances, such as proteolytic enzymes, reactive oxygen/nitrogen species, cytokines and chemokynes, which may be injurious to the adjacent endothelial cell and to the alveoli [39]. PMN apoptosis is a crucial injury-limiting mechanism of inflammatory resolution. Several inflammatory agents such as LPS, TNF, IL-8, IL-6, IL-1 and granulocyte colony stimulating factor (G-CSF) can delay apoptotic re‐ sponse, providing PMN with a longer life, allowing accumulating at local tissues [41]. NFκB has been reported as a modulator of apoptosis in inflammatory cells [42, 43] allowing a pro-

) T-cells can be categorized as

T cells) contributes to immunosup‐

ergize. T-cell subgroups are modified in sepsis. Helper (CD4<sup>+</sup>

634 Gene Therapy - Tools and Potential Applications

of circulating and tissue lymphocytes (B cells and CD4<sup>+</sup>

indirect ALI/ARDS is seen during sepsis.

inflammatory state.

α and lipopolysaccharide during sepsis may also induce apoptosis [35].

**3. Physiopathology of acute respiratory distress syndrome**

Gene therapy is defined as the introduction of nucleic acids into cells for the purpose of altering the course of a medical condition o disease [50]. In general, the advantages of gene therapy over the other treatments are the selective treatment of affected tissues, the possibility of using locally endogenous proteins in cases where its systemic application would incur in serious adverse secondary effects, and the possibility of therapeutic long term after a single application [51]. Currently, there are three categories of gene delivery methods: viral vector based, non-viral vector based and physical methods [52]. Viralbased gene delivery systems is accomplished by using replication-deficient viruses con‐ taining the gene of interest, but with the disease-causing sequences deleted from the viral genome [53] including RNA-based viral vectors [54, 55], DNA-based viral vectors such as adenoviral vectors [56], adeno-associated viruses (AAV) vectors orherpes simplex viral vector [57].The non-viral gene delivery methods use synthetic or natural compounds or physical forces to deliver a piece of DNA into a cell [58]. Two main groups of non-viral delivery methods have developed: chemical-based, including lipofection [59] and inorgan‐ ic nanoparticles that are usually prepared from metals, inorganic salts or ceramics [60]; and using physical forces such as local or rapid systemic injection [61], particle impact [62, 63], electric pulse [64] or laser irradiation [65].

#### **5. Gene therapy in sepsis**

Currently, there is evidence that applying therapeutic maneuvers such as early effective an‐ tibiotic administration, intensive fluid resuscitation, mechanical ventilation in selected pa‐ tients and use of C activated protein in sickest patients improve significantly the survive in these patients [66]. There are several clinical studies that are trying to validate another kind of therapies such as extra-renal depuration, levosimendan, the use of immunoglobulins, ni‐ tric oxide, statins, selenium, the use of enteral nutrition with eicosapentaenoic acid (EPA)/ψlinolenic acid (GLA) that are in progress [67]. Basic research and clinical trials have focused on alternative therapeutic approaches [68].

siRNA-987, 5'-CCCACUCGGAGAAGUUUAATT-3' against mCD14. *In vitro* experiments with RAW264.7 cells (a transformed murine macrophage cell line) shown that siRNA-224 ef‐ fectively inhibited LPS-induced TNFα, MIP-2 and IL-6 release and NO production [78].

Severely burned patients are greatly susceptible to infection with various pathogens [79]. Macrophages (MΦs) have an important role in antibacterial innate immunity. In methicillin-

converted by the TLRs stimulation and has the ability to kill bacteria, to produce reactive nitrogen intermediates, and to release antimicrobial peptides [80], playing a pivotal role in host microbial resistance. M2MΦ have reduced ability to kill bacteria; IL-10 and CCL7 re‐ leased by M2MΦ are inhibitory molecules on the pathogen-stimulated MΦ conversion to M1MΦ. IL-10 is also a deactivator of antibacterial immunocompetent cells [81] and an inhib‐ itory molecule on various immunocompetent cell functions. Asai et al have demonstrated that IL-10 antisense oligonucleotides in a severely burned mice prevents the burn associated conversion of MΦ to M2MΦ and infectious complications stemming for MRSA local infec‐

CCL2 is a chemokine that attracts and activates mononuclear cells. The necessity of this che‐ mokine for Th2-cell generation has been demonstrated. In a study Shigematsu K et al [83] tried to protect thermally injured mice orally infected with a lethal dose of *E. faecalis* by gene therapy utilizing phosphorothioate-CCL2 antisense oligodeoxynucleotides. They demon‐ strate that sepsis stemming from *E. faecalis* translocation in severely burned mice is control‐ lable by the gene therapy using CCL2 antisense ODNs, through the elimination of mesenteric lymph node macrophages (MLN- MΦ)-M2aMΦs and M2cMΦs subtypes. [83].

IL-1β binds the type-1 IL-1 receptor, while LPS binds to TLR4, both activates intracellular pathways by phosphorylation of IRAK family members including IRAK-1, which involve the MyD88 adaptor protein [84]. The group of Johns RE et al developed a family of "smart" polymeric carriers, termed encrypted polymers that enhance the cytoplasmic delivery of therapeutic antisense oligonucleotides (ASONs). This group has demonstrated that these ASONs block LPS activation of the transcription factor NFκB reducing the LPS-induced ex‐ pression of cytokines and chemokines. IL-6 shows a 2-fold decrease whereas TNFα expres‐ sion trended to decrease. There was a 2-fold decrease in expression of several genes

Caspases are pro-enzymes of the aspartate-specific cysteine protease family and its activa‐ tion plays a central role in the execution of apoptosis [86]. Depending of the stimuli, two cas‐ pase-activation pathways have been described, the mitochondria-initiated caspase-8 dependent pathway and mitochondria-initiated caspase-9-mediated pathway. Activation of these pathways initiates a downstream cascade of effector caspases, such as caspase-3 that

) and M2MΦ (IL-12- IL-10<sup>+</sup>

) differentiate in two dif‐

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 637

). The former are

resistant *Staphylococcus aureus* infection (MRSA), MΦs (IL-12- IL-10-

ferent subpopulations, M1MΦ (IL-12<sup>+</sup> IL-10-

including MCP1, MCP3, eotaxin and IP10 [85].

**5.2. Intracellular signaling**

tion did not develop [82].

**5.3. Apoptosis**

#### **5.1. Pattern associated membrane receptors**

Different approaches have designed trying to block the interaction between PAMs and PPRs. One is the generation of antibodies that bind TLRs. Studies conducted with anti-lipo‐ polysaccharide binding protein or anti-CD14 in experimental models of endotoxic shock and Gram-negative bacterial sepsis, failed to show a protection when treatment was admin‐ istered after LPS o simultaneously with or shortly after bacterial inoculation [69-71]. By us‐ ing a recombinant chimeric fusion protein composed of the N-terminal and central domains (amino-acids 1-334) of the extracellular part of TLR4 and the Fc portion of the human IgG1, Roger et al [72] produced an anti-TLR4 antibodies that inhibited LPS-induced intracellular signaling and cytokine production and protected mice from lethal endotoxic shock and E. coli bacterial sepsis, not only in pre-treatment with the antibodies, but also even when treat‐ ment was delayed for several hours after endotoxemia of the onset of sepsis.

The RAGEs (receptor for advanced glycation end products) are part of DAMPs that may play a role in the perpetuation of inflammation that carries to severe sepsis or septic shock. RAGEs are up-regulated in acute and chronic inflammation and bind multiple endogenous mediators involved in sepsis and products of oxidative stress [73]. In a recent work, Christa‐ ki et al demonstrated that blocking RAGEs either before or after infection protected mice from lethality in sepsis due to *S. pneumoniae* pneumonia [74] probably by indirect inhibition of NFκB activation.

Exposure to Staphylococcal enterotoxin (SE) or SE plus lipopolysaccharide (LPS, endotoxin) in mice, triggers vigorous intracellular signaling that leads to hyper-inflammation and re‐ lease of pro-inflammatory cytokines such as TNFα, INFγ, IL-1β, IL-1α, IL-2 and IL-6 by acti‐ vation of innate immunity [75]. In order to evaluate the role of MyD88, the anchor adaptor protein that integrates and transduces intracellular signals from TLRs and IL-1 receptor su‐ perfamily, Kisssner et al evaluates a synthetic molecule, hydrocinnamoyl-L-valyl-pyrroli‐ dine (Compd1), which mimics the BB-loop in the TIR domain of MyD88. They observed an inhibited pro-inflammatory cytokine production in human primary cells. Also, administra‐ tion of Compd1 to mice inhibited pro-inflammatory cytokine response and increased surviv‐ al from toxic shock induced death-limiting hyper-inflammation [76].

Recently, the knockdown or TLR2 by three different small interfering RNAs (siRNA) (A: 5' aactatccactggtgaaacaa-3', B: 5'-aaacttgtcagtggccagaaa-3', C: 5'-aaagtcttgattgattggcca-3') re‐ duce de tumorigenesis generated by the injection of BEL-7402 cells in an athymic mouse. Also, the levels of cytokines IL-6 and IL-8 were found to be markedly depressed [77]. In this line, Lei Ming et al have designed four siRNA:

siRNA-180, 5'-GCCUGGAAUACCUUCUAAATT-3'; siRNA-224, 5'-GGGCAGUUCACUGAUAUUATT-3'; siRNA-341, 5'-CAGGAACUGACUCUUGAAATT-3';

siRNA-987, 5'-CCCACUCGGAGAAGUUUAATT-3' against mCD14. *In vitro* experiments with RAW264.7 cells (a transformed murine macrophage cell line) shown that siRNA-224 ef‐ fectively inhibited LPS-induced TNFα, MIP-2 and IL-6 release and NO production [78].

#### **5.2. Intracellular signaling**

linolenic acid (GLA) that are in progress [67]. Basic research and clinical trials have focused

Different approaches have designed trying to block the interaction between PAMs and PPRs. One is the generation of antibodies that bind TLRs. Studies conducted with anti-lipo‐ polysaccharide binding protein or anti-CD14 in experimental models of endotoxic shock and Gram-negative bacterial sepsis, failed to show a protection when treatment was admin‐ istered after LPS o simultaneously with or shortly after bacterial inoculation [69-71]. By us‐ ing a recombinant chimeric fusion protein composed of the N-terminal and central domains (amino-acids 1-334) of the extracellular part of TLR4 and the Fc portion of the human IgG1, Roger et al [72] produced an anti-TLR4 antibodies that inhibited LPS-induced intracellular signaling and cytokine production and protected mice from lethal endotoxic shock and E. coli bacterial sepsis, not only in pre-treatment with the antibodies, but also even when treat‐

The RAGEs (receptor for advanced glycation end products) are part of DAMPs that may play a role in the perpetuation of inflammation that carries to severe sepsis or septic shock. RAGEs are up-regulated in acute and chronic inflammation and bind multiple endogenous mediators involved in sepsis and products of oxidative stress [73]. In a recent work, Christa‐ ki et al demonstrated that blocking RAGEs either before or after infection protected mice from lethality in sepsis due to *S. pneumoniae* pneumonia [74] probably by indirect inhibition

Exposure to Staphylococcal enterotoxin (SE) or SE plus lipopolysaccharide (LPS, endotoxin) in mice, triggers vigorous intracellular signaling that leads to hyper-inflammation and re‐ lease of pro-inflammatory cytokines such as TNFα, INFγ, IL-1β, IL-1α, IL-2 and IL-6 by acti‐ vation of innate immunity [75]. In order to evaluate the role of MyD88, the anchor adaptor protein that integrates and transduces intracellular signals from TLRs and IL-1 receptor su‐ perfamily, Kisssner et al evaluates a synthetic molecule, hydrocinnamoyl-L-valyl-pyrroli‐ dine (Compd1), which mimics the BB-loop in the TIR domain of MyD88. They observed an inhibited pro-inflammatory cytokine production in human primary cells. Also, administra‐ tion of Compd1 to mice inhibited pro-inflammatory cytokine response and increased surviv‐

Recently, the knockdown or TLR2 by three different small interfering RNAs (siRNA) (A: 5' aactatccactggtgaaacaa-3', B: 5'-aaacttgtcagtggccagaaa-3', C: 5'-aaagtcttgattgattggcca-3') re‐ duce de tumorigenesis generated by the injection of BEL-7402 cells in an athymic mouse. Also, the levels of cytokines IL-6 and IL-8 were found to be markedly depressed [77]. In this

ment was delayed for several hours after endotoxemia of the onset of sepsis.

al from toxic shock induced death-limiting hyper-inflammation [76].

line, Lei Ming et al have designed four siRNA:

siRNA-180, 5'-GCCUGGAAUACCUUCUAAATT-3';

siRNA-224, 5'-GGGCAGUUCACUGAUAUUATT-3';

siRNA-341, 5'-CAGGAACUGACUCUUGAAATT-3';

on alternative therapeutic approaches [68].

636 Gene Therapy - Tools and Potential Applications

**5.1. Pattern associated membrane receptors**

of NFκB activation.

Severely burned patients are greatly susceptible to infection with various pathogens [79]. Macrophages (MΦs) have an important role in antibacterial innate immunity. In methicillinresistant *Staphylococcus aureus* infection (MRSA), MΦs (IL-12- IL-10- ) differentiate in two dif‐ ferent subpopulations, M1MΦ (IL-12<sup>+</sup> IL-10- ) and M2MΦ (IL-12- IL-10<sup>+</sup> ). The former are converted by the TLRs stimulation and has the ability to kill bacteria, to produce reactive nitrogen intermediates, and to release antimicrobial peptides [80], playing a pivotal role in host microbial resistance. M2MΦ have reduced ability to kill bacteria; IL-10 and CCL7 re‐ leased by M2MΦ are inhibitory molecules on the pathogen-stimulated MΦ conversion to M1MΦ. IL-10 is also a deactivator of antibacterial immunocompetent cells [81] and an inhib‐ itory molecule on various immunocompetent cell functions. Asai et al have demonstrated that IL-10 antisense oligonucleotides in a severely burned mice prevents the burn associated conversion of MΦ to M2MΦ and infectious complications stemming for MRSA local infec‐ tion did not develop [82].

CCL2 is a chemokine that attracts and activates mononuclear cells. The necessity of this che‐ mokine for Th2-cell generation has been demonstrated. In a study Shigematsu K et al [83] tried to protect thermally injured mice orally infected with a lethal dose of *E. faecalis* by gene therapy utilizing phosphorothioate-CCL2 antisense oligodeoxynucleotides. They demon‐ strate that sepsis stemming from *E. faecalis* translocation in severely burned mice is control‐ lable by the gene therapy using CCL2 antisense ODNs, through the elimination of mesenteric lymph node macrophages (MLN- MΦ)-M2aMΦs and M2cMΦs subtypes. [83].

IL-1β binds the type-1 IL-1 receptor, while LPS binds to TLR4, both activates intracellular pathways by phosphorylation of IRAK family members including IRAK-1, which involve the MyD88 adaptor protein [84]. The group of Johns RE et al developed a family of "smart" polymeric carriers, termed encrypted polymers that enhance the cytoplasmic delivery of therapeutic antisense oligonucleotides (ASONs). This group has demonstrated that these ASONs block LPS activation of the transcription factor NFκB reducing the LPS-induced ex‐ pression of cytokines and chemokines. IL-6 shows a 2-fold decrease whereas TNFα expres‐ sion trended to decrease. There was a 2-fold decrease in expression of several genes including MCP1, MCP3, eotaxin and IP10 [85].

#### **5.3. Apoptosis**

Caspases are pro-enzymes of the aspartate-specific cysteine protease family and its activa‐ tion plays a central role in the execution of apoptosis [86]. Depending of the stimuli, two cas‐ pase-activation pathways have been described, the mitochondria-initiated caspase-8 dependent pathway and mitochondria-initiated caspase-9-mediated pathway. Activation of these pathways initiates a downstream cascade of effector caspases, such as caspase-3 that cleaves substrates such as D4-GDI leading to cell death [87]. The group of Ayala A et al in 2005 demonstrated that suppression of Fas or caspase-8 gene expression with hydrodynamic administration of siRNA conferred a survival advantage in septic mice model after caecal ligation and perforation (CLP) [88]. In a work of Matsuda N et al, they examined the thera‐ peutic efficacy of caspase-8 and caspase-3 gene silencing with siRNAs delivered by systemic injection in a CLP endotoxic shock mouse model. They demonstrate that *in vivo* delivery of caspase-8/caspase-3 siRNAs conferred a dramatic survival advantage to CLP mice as com‐ pared to controls. Also they demonstrated that the survival benefit was observed despite ad‐ ministration of siRNA as late as 10h after CLP [88].

exudate floods the alveolar spaces, impairs gas exchange and precipitates respiratory failure [92]. Several studies indicate that CLP (cecal ligation and puncture) sepsis model, sepsis and endotoxemia impair the expression of heat shock protein (HSP-70). Data shown that HSP-70 can limit inflammatory responses protect proteins from damage, restore function to proteins that are damaged and prevent cellular destruction, key processes of ALI/ARDS [93]. Weiss et al have demonstrated that the use of an adenoviral vector (AdHSP, an adenovirus carry‐ ing the gene for HSP-70) correcting the relative defect in HSP-70 expression prevents neutro‐ phil accumulation, reduce protein rich edema fluid and improve the outcome in ARDS

Injury of the alveolo-capillary barrier alters active Na+ transport, leading to impaired edema fluid clearance from the alveolar spaces. Failure to return to normal clearance is associated with poor prognosis [95]. The primary force driving fluid reabsorption from the alveolar space into the interstitium and the pulmonary circulation is active Na+ transport. Sodium is taken up on the apical surface of the alveolar epithelium by amiloride-sensitive and -insensi‐

channels [96] and is subsequently pumped out of the cell by the Na+

the β1-subunit gene by utilizing a replication-incompetent human type-5 adenovirus ex‐

using electroporation increased alveolar fluid reabsorption [98]. Furthermore, while rats ex‐ posed to 100% oxygen develop ALI and impaired alveolar fluid clearance; overexpression of

100% survival over 14 days of hyperoxia (compared with 25-31% survival in the non-treated

In this line, Stern M et al used a cationic liposome to transfer cDNA encoding both α and β

thiourea; they observe a significant resolution of pulmonary edema *in vivo*. Also, overex‐ pression of the β2-adrenergic receptor leads to increased alveolar fluid clearance in rats by

ATPase function, probably enhancing responsiveness to endogenous catecholamines in the

The regulation of alveolar transport proteins is vital in the maintenance of alveolar fluid bal‐

and protein abundance at the plasma membrane by promoting the endocytosis of the pump, which contributes to a decrease in alveolar fluid reabsorption in both *in vivo* an *ex vivo* mod‐ els of hypoxia. Also, the overexpression of the reactive oxygen species scavenger, SOD2,

/K+

/K+

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 639

/K+

/K+

/K+ -

/K+

secondary to CLP [94].

triphosphatase (Na+

ed the importance of Na+

/K+

/K+

or null virus-treated control groups) [99].

/K+

alveolar epithelium [100].

/K+

ance and, most importantly, overexpression of the Na+

increasing both membrane-bound amiloride-sensitive Na+

ance in patients [101]. The exposure to hypoxia results in decreased Na+

prevents this hypoxia-mediated decrease in alveolar fluid reabsorption and Na+

fold compared with controls [97]. Similarly, gene transfer of the Na+

tive Na+

pressing Na+

/K+

subunits of Na+

function [102].

the Na+

BRCA1 is a critical regulator of DNA damage repair and cell survival. In a recent article, Teoh H et al demonstrated a reduction in 24 hours post caecal ligation and perforation and thioglycollate stimulation mortality with pretreatment with human BRCA1 adenovirus (AdBRCA1). Treatment with AdBRCA1, a human adenovirus type-5 (dE1/E3), blunted CLPassociated cardiac, pulmonary, hepatic and renal dysfunction and also reduced CLP-elicited double strand breaks and apoptosis in the liver. BRCA1 gene therapy was associated with lower CLP-evoked cardiac and hepatic superoxide generation that in the liver was in part due to improved reactive oxygen species removal. CLP also elevated mesenteric arteriolar and serum intercellular adhesion molecule-1, both of which were partially abrogated with AdBRCA1 administration. Thioglycollate-challenged AdBRCA1-treated mice displayed re‐ duced peritoneal neutrophil recruitment and dampened cytokine elaboration relative to their Ad-null-treated counterparts [89].

#### **6. Gene therapy in ARDS/ALI**

Over the past 20 years, the feasibility of using gene transfer to treat ALI/ARDS has been demonstrated using a variety of viral and non-viral vectors to deliver various transgenes to the lung [90].

#### **6.1. Strategies to increase pulmonary surfactant**

ALI/ARDS is a surfactant-deficient state. *Pseudomonas aeruginosa* infection is a cause of pul‐ monary infection and ARDS with surfactant deficient phenotype. Zhou J et al have demon‐ strated the attenuation of the deleterious effects of *Pseudomonas aeruginosa* infection by adenoviral gene transfer overexpressing CCTpenta (a mutant form of the regulatory enzyme CCTα required for the biosynthesis of dipalmitoyl phosphatidylcholine (DPPC), the major phospholipid of surfactant) with a significant increase of the biosynthesis of surfactant. This study suggests that augmentation of DPPC synthesis via gene delivery of CCTα can attenu‐ ate impaired lung function in surfactant-deficient states such as bacterial sepsis [91].

#### **6.2. Strategies to improve pulmonary edema**

The physiological hallmark of ARDS is disruption of the alveolar-capillary membrane barri‐ er, leading to development of non-cardiogenic pulmonary edema, in which proteinaceous exudate floods the alveolar spaces, impairs gas exchange and precipitates respiratory failure [92]. Several studies indicate that CLP (cecal ligation and puncture) sepsis model, sepsis and endotoxemia impair the expression of heat shock protein (HSP-70). Data shown that HSP-70 can limit inflammatory responses protect proteins from damage, restore function to proteins that are damaged and prevent cellular destruction, key processes of ALI/ARDS [93]. Weiss et al have demonstrated that the use of an adenoviral vector (AdHSP, an adenovirus carry‐ ing the gene for HSP-70) correcting the relative defect in HSP-70 expression prevents neutro‐ phil accumulation, reduce protein rich edema fluid and improve the outcome in ARDS secondary to CLP [94].

cleaves substrates such as D4-GDI leading to cell death [87]. The group of Ayala A et al in 2005 demonstrated that suppression of Fas or caspase-8 gene expression with hydrodynamic administration of siRNA conferred a survival advantage in septic mice model after caecal ligation and perforation (CLP) [88]. In a work of Matsuda N et al, they examined the thera‐ peutic efficacy of caspase-8 and caspase-3 gene silencing with siRNAs delivered by systemic injection in a CLP endotoxic shock mouse model. They demonstrate that *in vivo* delivery of caspase-8/caspase-3 siRNAs conferred a dramatic survival advantage to CLP mice as com‐ pared to controls. Also they demonstrated that the survival benefit was observed despite ad‐

BRCA1 is a critical regulator of DNA damage repair and cell survival. In a recent article, Teoh H et al demonstrated a reduction in 24 hours post caecal ligation and perforation and thioglycollate stimulation mortality with pretreatment with human BRCA1 adenovirus (AdBRCA1). Treatment with AdBRCA1, a human adenovirus type-5 (dE1/E3), blunted CLPassociated cardiac, pulmonary, hepatic and renal dysfunction and also reduced CLP-elicited double strand breaks and apoptosis in the liver. BRCA1 gene therapy was associated with lower CLP-evoked cardiac and hepatic superoxide generation that in the liver was in part due to improved reactive oxygen species removal. CLP also elevated mesenteric arteriolar and serum intercellular adhesion molecule-1, both of which were partially abrogated with AdBRCA1 administration. Thioglycollate-challenged AdBRCA1-treated mice displayed re‐ duced peritoneal neutrophil recruitment and dampened cytokine elaboration relative to

Over the past 20 years, the feasibility of using gene transfer to treat ALI/ARDS has been demonstrated using a variety of viral and non-viral vectors to deliver various transgenes to

ALI/ARDS is a surfactant-deficient state. *Pseudomonas aeruginosa* infection is a cause of pul‐ monary infection and ARDS with surfactant deficient phenotype. Zhou J et al have demon‐ strated the attenuation of the deleterious effects of *Pseudomonas aeruginosa* infection by adenoviral gene transfer overexpressing CCTpenta (a mutant form of the regulatory enzyme CCTα required for the biosynthesis of dipalmitoyl phosphatidylcholine (DPPC), the major phospholipid of surfactant) with a significant increase of the biosynthesis of surfactant. This study suggests that augmentation of DPPC synthesis via gene delivery of CCTα can attenu‐

The physiological hallmark of ARDS is disruption of the alveolar-capillary membrane barri‐ er, leading to development of non-cardiogenic pulmonary edema, in which proteinaceous

ate impaired lung function in surfactant-deficient states such as bacterial sepsis [91].

ministration of siRNA as late as 10h after CLP [88].

638 Gene Therapy - Tools and Potential Applications

their Ad-null-treated counterparts [89].

**6. Gene therapy in ARDS/ALI**

**6.1. Strategies to increase pulmonary surfactant**

**6.2. Strategies to improve pulmonary edema**

the lung [90].

Injury of the alveolo-capillary barrier alters active Na+ transport, leading to impaired edema fluid clearance from the alveolar spaces. Failure to return to normal clearance is associated with poor prognosis [95]. The primary force driving fluid reabsorption from the alveolar space into the interstitium and the pulmonary circulation is active Na+ transport. Sodium is taken up on the apical surface of the alveolar epithelium by amiloride-sensitive and -insensi‐ tive Na+ channels [96] and is subsequently pumped out of the cell by the Na+ /K+ -adenosine triphosphatase (Na+ /K+ -ATPase) on the baso-lateral side [96]. Some studies have demonstrat‐ ed the importance of Na+ /K+ -ATPase in ALI/ARDS. In normal adults rats, overexpression of the β1-subunit gene by utilizing a replication-incompetent human type-5 adenovirus ex‐ pressing Na+ /K+ -ATPase-β1 subunit cDNA increased alveolar edema clearance over twofold compared with controls [97]. Similarly, gene transfer of the Na+ /K+ -ATPase-β1 subunit using electroporation increased alveolar fluid reabsorption [98]. Furthermore, while rats ex‐ posed to 100% oxygen develop ALI and impaired alveolar fluid clearance; overexpression of the Na+ /K+ -ATPase-β1 subunit in the alveolar epithelium of rats increased lung liquid clear‐ ance and, most importantly, overexpression of the Na+ /K+ -ATPase-β1 subunit resulted in 100% survival over 14 days of hyperoxia (compared with 25-31% survival in the non-treated or null virus-treated control groups) [99].

In this line, Stern M et al used a cationic liposome to transfer cDNA encoding both α and β subunits of Na+ /K+ -ATPase to the lung of a mouse model of pulmonary edema induced by thiourea; they observe a significant resolution of pulmonary edema *in vivo*. Also, overex‐ pression of the β2-adrenergic receptor leads to increased alveolar fluid clearance in rats by increasing both membrane-bound amiloride-sensitive Na+ -channel expression and Na+ /K+ - ATPase function, probably enhancing responsiveness to endogenous catecholamines in the alveolar epithelium [100].

The regulation of alveolar transport proteins is vital in the maintenance of alveolar fluid bal‐ ance in patients [101]. The exposure to hypoxia results in decreased Na+ /K+ -ATPase activity and protein abundance at the plasma membrane by promoting the endocytosis of the pump, which contributes to a decrease in alveolar fluid reabsorption in both *in vivo* an *ex vivo* mod‐ els of hypoxia. Also, the overexpression of the reactive oxygen species scavenger, SOD2, prevents this hypoxia-mediated decrease in alveolar fluid reabsorption and Na+ /K+ -ATPase function [102].

#### **6.3. Strategies to afford oxidant injury-related injury, apoptosis and inflammation**

Keratinocyte growth factor (KGF) is an epithelial-specific growth factor secreted by fibro‐ blast and vascular smooth muscle cells and a main mitogen for alveolar type II cells [103]. Baba et al have demonstrated that transient over-expression of KGF in the lungs attenuate pathophysiological impairments in hyperoxia-induced acute lung injury by increasing Ki67 and surfactant protein C (Sp-C)-positive cells and proliferation of epithelial cuboidal cells [104]. There is an abundance of evidence regarding the protective effect of pre-treatment with KGF on lung injury induced by hyperoxia, acid instillation, radiation, bleomycin, αnaphthylthiourea, ventilator and bacterial pneumonia there are some studies that supports the potential clinical application of KGF-2 in the treatment of ALI/ARDS [105].

hyperoxia models and have shown significant reductions in inflammation and subsequent

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 641

Sepsis and acute lung injury/acute respiratory distress syndrome are important pathologies in critical care medicine. There are increasing evidence from relevant pre-clinical studies that support the efficacy of gene-based therapies. Multiple barriers exist to the successful use of gene therapy in critical care medicine and particularly in sepsis and ALI/ARDS. Fu‐ ture research approaches are necessary to overcome these barriers by developing better viral and non-viral vectors, enhanced and specific gene expression strategies, improved cellular

Although the treatment by transference of genetic material still presents many challenges, the technology is rapidly evolving and the possible use in clinical trials could be in a near future. So, the aim of this chapter was to understand the molecular mechanisms involved in acute respiratory distress syndrome and sepsis, to review the viral and non-viral gene thera‐ pies that have been developed to improve survival and to address the challenges of gene

This work was supported by CONACYT grant 164413 and PAPIIT-UNAM grant IN204410.

and Angel Zarain-Herzberg2

1 Intensive Care Unit, Hospital Universitari de Bellvitge, L'Hospitalet Llobregat, Barcelona,

2 Biochemistry Department, School of Medicine, Universidad Nacional Autónoma de Méxi‐

[1] Vincent JL. Evidence-based medicine in the ICU: important advances and limitations.

Chest. 2004;126(2):592-600. Epub 2004/08/11.

therapy in critical care patients using these two life-threating conditions as a model.

lung injury [90].

**Acknowledgments**

**Author details**

Spain

Gabriel J. Moreno-González1

co, Mexico City, México

**References**

**7. Future directions and conclusion**

uptake of vectors and better therapeutic targets.

Human angiopoeitin-1 (ANGPT1), a ligand for the endothelial-restricted receptor TEK tyro‐ sine kinase, plays an essential role in blood vessel maturation and stabilization during em‐ bryonic development. In postnatal, ANGPT1 maintains the normal quiescent phenotype of vascular ECs, protecting against vascular inflammation reducing permeability and promot‐ ing ECs survival. In a study of Mei SH and co-workers carried out in an ALI mice model (by intra-tracheal instillation of LPS), they have demonstrated that mesenchymal stem cells (MSCs) administration alone into the pulmonary circulation partially prevents LPS-induced lung inflammation. However, cell-based gene transfer using pANGPT1-transfected MSCs resulted in further improvement in both alveolar inflammation and membrane permeability. Also, MSCs-pANGPT1 dramatically reduced cytokine levels (IFNγ, TNFα, IL-6 and IL1-β) to the baseline values observed in naïve mice, suggesting a potential therapeutic approach to ALI/ARDS [106].

Pearl M and colleagues in a 2005 study using Fas- and caspase-8 siRNA intra-tracheal ad‐ ministration in a CLP mice model of sepsis demonstrated that the main targets of siRNA de‐ livery are the epithelial cells. Also, that down-regulation of Fas but not caspase-8 reduces pulmonary apoptosis and lung inflammation, decreases neutrophil influx and attenuates ALI [107].

Overexpression of interleukin IL-10 trough recombinant adeno-associated virus type-5 (AAV5) vector expressing murine IL-10 into pulmonary, tissue proinflammatory cytokines IL-1β and TNFα, macrophage inhibitory protein-1αand keratinocyte chemoattractant in the epithelial lining fluid and lung homogenate were decreased and neutrophil infiltration was less pronounced and more localized neutrophil infiltration in lung section [108].

Finally, Hemoxygenase-1 (HO-1) is an inducible isoform of the first and rate-controlling en‐ zyme of the degradation of heme into iron, carbon monoxide, and biliverdin, the latter being subsequently converted into bilirubin. Several positive biological effects exerted by this en‐ zyme have gained attention, as anti-inflammatory, antiapoptotic, angiogenic, and cytopro‐ tective functions are attributable to carbon monoxide and/or bilirubin Also, the enzyme has been involved in controlling infiltration of neutrophils into the injured lung and in the reso‐ lution of inflammation by modulating apoptotic cell death and cytokine expression. Several groups have delivered HO-1 expressing adenoviruses to the lungs in both pneumonia and hyperoxia models and have shown significant reductions in inflammation and subsequent lung injury [90].

#### **7. Future directions and conclusion**

**6.3. Strategies to afford oxidant injury-related injury, apoptosis and inflammation**

the potential clinical application of KGF-2 in the treatment of ALI/ARDS [105].

to ALI/ARDS [106].

640 Gene Therapy - Tools and Potential Applications

ALI [107].

Keratinocyte growth factor (KGF) is an epithelial-specific growth factor secreted by fibro‐ blast and vascular smooth muscle cells and a main mitogen for alveolar type II cells [103]. Baba et al have demonstrated that transient over-expression of KGF in the lungs attenuate pathophysiological impairments in hyperoxia-induced acute lung injury by increasing Ki67 and surfactant protein C (Sp-C)-positive cells and proliferation of epithelial cuboidal cells [104]. There is an abundance of evidence regarding the protective effect of pre-treatment with KGF on lung injury induced by hyperoxia, acid instillation, radiation, bleomycin, αnaphthylthiourea, ventilator and bacterial pneumonia there are some studies that supports

Human angiopoeitin-1 (ANGPT1), a ligand for the endothelial-restricted receptor TEK tyro‐ sine kinase, plays an essential role in blood vessel maturation and stabilization during em‐ bryonic development. In postnatal, ANGPT1 maintains the normal quiescent phenotype of vascular ECs, protecting against vascular inflammation reducing permeability and promot‐ ing ECs survival. In a study of Mei SH and co-workers carried out in an ALI mice model (by intra-tracheal instillation of LPS), they have demonstrated that mesenchymal stem cells (MSCs) administration alone into the pulmonary circulation partially prevents LPS-induced lung inflammation. However, cell-based gene transfer using pANGPT1-transfected MSCs resulted in further improvement in both alveolar inflammation and membrane permeability. Also, MSCs-pANGPT1 dramatically reduced cytokine levels (IFNγ, TNFα, IL-6 and IL1-β) to the baseline values observed in naïve mice, suggesting a potential therapeutic approach

Pearl M and colleagues in a 2005 study using Fas- and caspase-8 siRNA intra-tracheal ad‐ ministration in a CLP mice model of sepsis demonstrated that the main targets of siRNA de‐ livery are the epithelial cells. Also, that down-regulation of Fas but not caspase-8 reduces pulmonary apoptosis and lung inflammation, decreases neutrophil influx and attenuates

Overexpression of interleukin IL-10 trough recombinant adeno-associated virus type-5 (AAV5) vector expressing murine IL-10 into pulmonary, tissue proinflammatory cytokines IL-1β and TNFα, macrophage inhibitory protein-1αand keratinocyte chemoattractant in the epithelial lining fluid and lung homogenate were decreased and neutrophil infiltration was

Finally, Hemoxygenase-1 (HO-1) is an inducible isoform of the first and rate-controlling en‐ zyme of the degradation of heme into iron, carbon monoxide, and biliverdin, the latter being subsequently converted into bilirubin. Several positive biological effects exerted by this en‐ zyme have gained attention, as anti-inflammatory, antiapoptotic, angiogenic, and cytopro‐ tective functions are attributable to carbon monoxide and/or bilirubin Also, the enzyme has been involved in controlling infiltration of neutrophils into the injured lung and in the reso‐ lution of inflammation by modulating apoptotic cell death and cytokine expression. Several groups have delivered HO-1 expressing adenoviruses to the lungs in both pneumonia and

less pronounced and more localized neutrophil infiltration in lung section [108].

Sepsis and acute lung injury/acute respiratory distress syndrome are important pathologies in critical care medicine. There are increasing evidence from relevant pre-clinical studies that support the efficacy of gene-based therapies. Multiple barriers exist to the successful use of gene therapy in critical care medicine and particularly in sepsis and ALI/ARDS. Fu‐ ture research approaches are necessary to overcome these barriers by developing better viral and non-viral vectors, enhanced and specific gene expression strategies, improved cellular uptake of vectors and better therapeutic targets.

Although the treatment by transference of genetic material still presents many challenges, the technology is rapidly evolving and the possible use in clinical trials could be in a near future. So, the aim of this chapter was to understand the molecular mechanisms involved in acute respiratory distress syndrome and sepsis, to review the viral and non-viral gene thera‐ pies that have been developed to improve survival and to address the challenges of gene therapy in critical care patients using these two life-threating conditions as a model.

#### **Acknowledgments**

This work was supported by CONACYT grant 164413 and PAPIIT-UNAM grant IN204410.

#### **Author details**

Gabriel J. Moreno-González1 and Angel Zarain-Herzberg2

1 Intensive Care Unit, Hospital Universitari de Bellvitge, L'Hospitalet Llobregat, Barcelona, Spain

2 Biochemistry Department, School of Medicine, Universidad Nacional Autónoma de Méxi‐ co, Mexico City, México

#### **References**

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[15] Villar J, Perez-Mendez L, Lopez J, Belda J, Blanco J, Saralegui I, et al. An early PEEP/ FIO2 trial identifies different degrees of lung injury in patients with acute respiratory distress syndrome. American journal of respiratory and critical care medicine.

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 643

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[17] Martin GS. Temporal changes in clinical outcomes with ARDS. Chest. 2005;128(2):

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[19] Rubenfeld GD. Epidemiology of acute lung injury. Crit Care Med. 2003;31(4

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**Chapter 27**

**Clinical and Translational Challenges in**

Divya Pankajakshan and Devendra K. Agrawal

Additional information is available at the end of the chapter

apeutic neo-vascularization to counteract ischemia.

http://dx.doi.org/10.5772/54075

**1. Introduction**

**Gene Therapy of Cardiovascular Diseases**

Cardiovascular (CV) disease is the most prevalent life-threatening clinical problem and is a major cause of disability and economic burden worldwide [1]. Despite extensive phar‐ macotherapies, there remain many vascular conditions for which pharmacological inter‐ ventions are either non-existent or largely ineffective. CV gene therapy offers the benefit of sustained and/or controlled expression of desired proteins in cell types, which makes it more beneficial in providing durable clinical benefits [2]. The therapeutic gene works by either over-expressing therapeutically beneficial proteins, replacing a deficient gene or its expression proteins, or silencing a particular gene whose expression is not beneficial in the clinical scenario [3]. In addition, success of gene therapy also depends on the choice of the vector and the delivery approach. Blood vessels are among the most feasible tar‐ gets for gene therapy because of ease of access using a catheter or by systemic delivery. The new genetic material should enter the cells in the vasculature overcoming the ana‐ tomical, cellular and physiological barriers and induce the expression of the transfected gene in the target tissue. The target cells in the arteries are endothelial cells (EC), smooth muscle cells (SMC) and fibroblasts, which constitute the intimal, medial and adventitial layers, respectively [4]. In the case of atherosclerotic lesions, macrophages also become a target cell. For the treatment of cardiovascular diseases, gene therapy strategies have been designed to enhance re-endothelialization and EC function to reduce thrombosis, inhibit SMC proliferation and migration to prevent neointimal hyperplasia, and to improve ther‐

Viral and non-viral vector systems have been evaluated for gene transfer to the vasculature. Lipoplexes, polyplexes and lipopolyplexes as well as naked DNA have been used as nonviral vectors for gene delivery to vascular tissues. Retroviruses, lentiviruses, adenoviruses

> © 2013 Pankajakshan and Agrawal; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases**

Divya Pankajakshan and Devendra K. Agrawal

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54075

#### **1. Introduction**

[105] Fang X, Bai C, Wang X. Potential clinical application of KGF-2 (FGF-10) for acute lung injury/acute respiratory distress syndrome. Expert review of clinical pharmacol‐

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Cardiovascular (CV) disease is the most prevalent life-threatening clinical problem and is a major cause of disability and economic burden worldwide [1]. Despite extensive phar‐ macotherapies, there remain many vascular conditions for which pharmacological inter‐ ventions are either non-existent or largely ineffective. CV gene therapy offers the benefit of sustained and/or controlled expression of desired proteins in cell types, which makes it more beneficial in providing durable clinical benefits [2]. The therapeutic gene works by either over-expressing therapeutically beneficial proteins, replacing a deficient gene or its expression proteins, or silencing a particular gene whose expression is not beneficial in the clinical scenario [3]. In addition, success of gene therapy also depends on the choice of the vector and the delivery approach. Blood vessels are among the most feasible tar‐ gets for gene therapy because of ease of access using a catheter or by systemic delivery. The new genetic material should enter the cells in the vasculature overcoming the ana‐ tomical, cellular and physiological barriers and induce the expression of the transfected gene in the target tissue. The target cells in the arteries are endothelial cells (EC), smooth muscle cells (SMC) and fibroblasts, which constitute the intimal, medial and adventitial layers, respectively [4]. In the case of atherosclerotic lesions, macrophages also become a target cell. For the treatment of cardiovascular diseases, gene therapy strategies have been designed to enhance re-endothelialization and EC function to reduce thrombosis, inhibit SMC proliferation and migration to prevent neointimal hyperplasia, and to improve ther‐ apeutic neo-vascularization to counteract ischemia.

Viral and non-viral vector systems have been evaluated for gene transfer to the vasculature. Lipoplexes, polyplexes and lipopolyplexes as well as naked DNA have been used as nonviral vectors for gene delivery to vascular tissues. Retroviruses, lentiviruses, adenoviruses

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and adeno-associated viruses have been tested as viral vectors. Both systems have their own advantages and disadvantages that determine its use for a particular subset of CV diseases. Another challenge is the development of delivery approaches that are clinically viable and are capable of achieving consistent therapy for diseased arterial tissues. The efficiency of lo‐ calization, restriction of systemic distribution and adequacy of permeation into the target tissue are required for the optimal delivery of the vector. It is also dependent on the require‐ ments of a given patho-physiological situation. Systemic, intravascular and perivascular ap‐ proaches are used for gene delivery to the vasculature.

tors demonstrate significantly broadened tropism and high stability and have been used to demonstrate efficient transgene delivery *in vitro* into SMCs and ECs from human sa‐ phenous vein [10], human coronary artery SMCs and ECs [11], and cardiomyocytes [12].

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

http://dx.doi.org/10.5772/54075

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Ad vectors are the most commonly used viral vectors in the CV system. They transfect non-dividing cells efficiently [Figure 1], but sustained gene expression is limited to ap‐ proximately 2 weeks because the gene is kept episomal [2]. The administration of the Ad vectors is almost invariably associated with the development of systemic neutralizing an‐ tibodies directed against the vector [13]. Therefore, lowering the immunogenicity of the Ad virus is desirable and can be achieved by deleting genes that encode viral proteins [14]. Another method of reducing the inflammatory reaction to gene transfer by Ad vec‐ tors is to preserve the E3 region, which is supposed to modulate the host immune re‐ sponse *in vivo* [15]. When systemically administered, Ad5 poorly transduced ECs but could effectively transduce medial SMCs during endothelial denudation [5]. Efficient my‐ ocardial transduction was observed following local delivery of Ad5 vectors in porcine

AAV vectors have emerged as versatile vehicles for gene delivery due to their efficient in‐ fection of dividing and non-dividing cells in the presence of helper virus, sustained main‐ tenance of viral genome leading to long-term expression of the transgene, and a strong clinical safety profile [17]. AAV is non-pathogenic since it cannot replicate without the as‐ sistance of a helper virus. Recombinant AAV (rAAV) vectors have almost the entire viral genome removed, thereby yielding a delivery vehicle with enhanced safety and reduced immunogenicity [18]. The AAV *Rep* and *Cap* genes, which are required for viral replica‐ tion and packaging, are supplied by a helper plasmid during the production process. Wild type AAV preferentially integrates to a specific locus of human chromosome 19. The rAAV has mechanisms for sustained episomal maintenance or semi-randomly integrates at a low rate [19]. Problems with AAV vectors include limited tissue tropism for sero‐ types that bind heparan sulphate, challenges with preexisting immunity due to prior ex‐ posure, and also substantially delayed onset of transgene expression compared to other

Even though the transfection efficiency of non-viral vectors are lower than that of their viral counterparts, they are associated with many advantages such as low immunogenic re‐ sponse, the capacity to carry large inserts of DNA (52Kb), the possibility of selective modifi‐ cation using ligand and large scale manufacture [20]. Ideal non-viral vectors should be degradable into low molecular weight components in response to biological stimuli for low‐ er toxicity and effective systemic clearance. They should also be efficient in overcoming ex‐ tracellular and intracellular barriers and tissue/cell-targeted for specific accumulations [21]. In this group of vectors, naked DNA, cationic liposomes and cationic polymers have been

heart, where almost 80% of cardiomyocytes were transduced [16].

vectors.

**2.2. Non-viral vectors**

used for vascular gene transfer.

In this chapter, our goal is to summarize the current understanding of gene therapy strat‐ egies used to treat CV diseases, specifically the therapies targeting thrombosis, atherogene‐ sis, SMC proliferation and migration, modification of extracellular matrix (ECM) and regeneration of the endothelial cell layer. We will discuss various vectors and delivery ap‐ proaches used in the CV gene therapy and describe, in detail, the challenges associated with each approach.

#### **2. Vectors in vascular gene therapy**

The ideal vector for clinical application would target the specific cell, offer the capacity to transfer large DNA sequences, result in therapeutic levels of transgene expression that are not attenuated by the host immune response, express transgene for a duration required to alleviate the clinical problem, pose no risk of toxicity either acutely (as a result of immuno‐ genicity or unregulated transgene expression) or in the long-term (such as oncogenesis), and be cost-effective and easy to produce in therapeutically applicable quantity [5]. Currently, no available vector fulfils all these criteria; therefore, a perfect vector for vascular gene thera‐ py does not exist. Nonetheless, viral and non-viral vector systems have been evaluated for gene transfer to the vasculature.

#### **2.1. Viral vectors**

Retroviruses, adenoviruses (Ad) and adeno-associated viruses (AAV) are used as viral vectors in vascular gene transfer. Recombinant retroviruses are RNA viruses that are ca‐ pable of integrating transgene into the target genome. Disadvantages of this vector in‐ clude instability, the requirement of cell division for gene transfer and the inability to attain high titers. Since the majority of vascular cells are not undergoing mitosis at the time of exposure to the viral vector, the efficiency of gene delivery to vascular cells by such vectors may be as low as 1% to 2% [6]. Attempts have been made to increase the transduction efficiency in endothelial cell using multiple viral exposures [7] or increasing viral titers by ultracentrifugation [8]. Murine leukemia retroviral vectors (MuLV) pseudo‐ typed with the vesicular stomatitis virus G glycoprotein (VSV-G) have the capacity to transfect human ECs and SMCs *in vitro* with significant improvement in stability and transduction efficiency [9]. Unlike other retroviruses, lentiviruses are able to transduce non-dividing cells, which is an attractive characteristic for CV gene therapy. These vec‐ tors demonstrate significantly broadened tropism and high stability and have been used to demonstrate efficient transgene delivery *in vitro* into SMCs and ECs from human sa‐ phenous vein [10], human coronary artery SMCs and ECs [11], and cardiomyocytes [12].

Ad vectors are the most commonly used viral vectors in the CV system. They transfect non-dividing cells efficiently [Figure 1], but sustained gene expression is limited to ap‐ proximately 2 weeks because the gene is kept episomal [2]. The administration of the Ad vectors is almost invariably associated with the development of systemic neutralizing an‐ tibodies directed against the vector [13]. Therefore, lowering the immunogenicity of the Ad virus is desirable and can be achieved by deleting genes that encode viral proteins [14]. Another method of reducing the inflammatory reaction to gene transfer by Ad vec‐ tors is to preserve the E3 region, which is supposed to modulate the host immune re‐ sponse *in vivo* [15]. When systemically administered, Ad5 poorly transduced ECs but could effectively transduce medial SMCs during endothelial denudation [5]. Efficient my‐ ocardial transduction was observed following local delivery of Ad5 vectors in porcine heart, where almost 80% of cardiomyocytes were transduced [16].