*3.2.4. Herpes Simplex Virus Vectors (HSV)*

and minus strands packaged with equal efficiency (Daly, 2003). No human disease has been associated with AAV infection which is an important feature when thinking in AAV as gene vectors. Six serotypes of AAV have been described with AAV2 being the most widely used for gene-transfer studies (Hermonat et al., 1984). AAV2 cell entry is mediated by binding to heparin sulfate proteoglycans and αvβ5 integrin; fibroblast growth factor receptor-1 (FGFR-1) may also be involved. The distribution of these molecules on many different cell types can explain the prolonged *in vivo* expression following AAV treatment seen in the liver, brain, skeletal muscle, lung, and hematopoietic stem cells of animal models (Daly, 2003). AAV vectors contain no viral genes that could elicit undesirable immune or inflammatory responses. The primary host reaction that might have an unwanted impact is the production of neutralizing

174 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

One major concern when developing AAV vectors for gene delivery is that DNA constructs larger than the wild-type 4.7 kb sequence do not package well and vector titers decrease sharply thus constituting an insertional limitation for the cDNAs. Another important issue is the frequently seen integration of AAV genome in the host cell chromosomes. However, the available evidence indicates that integration of wild type AAV *in vivo* does not reflect the experimental *in vitro* observations, but appears to be a rather rare event and AAV genomes mostly persist as episomes, as has also been demonstrated for AAV vectors (Carter et al., 2009).

Retroviruses are lipid-enveloped viruses; with nucleocapsids containing two copies of a linear, positive-stranded 7–11 kb RNA genome. The family *Retroviridae* contains various viruses that have shown potential utility for gene therapy, such as the **gammaretrovirus** *(or simple retrovi‐ rus)*, *spumaviruses* and **lentiviruses** *(or complex retrovirus)*. Following attachment and receptormediated entry into host cells, viral reverse transcriptase and integrase enzymes mediate reverse transcription and integration of the virus genome into the host-cell chromatin. Retroviral vectors have the ability for stable integration and allow long-term expression so that theoretically a single administration could have a sustained, potentially even, lifelong curative effect (Schambach et al., 2009). As for any viral vector, replication-deficiency is a condition. To achieve this goal, the retroviral coding sequences have to be removed, which creates at least 6 kb space for the transgene of interest. Since neither structural proteins nor replication enzymes are encoded by the target cell, the generation of replication-competent virus is prevented. The gammaretroviruses cannot infect quiescent, non-dividing cells, which is a handicap of the vectors derived from these retroviruses. However, this can be overcome by the use of lentiviral

For most RV, taking advantage of the insertional mechanism, the simplest application is in the production of cell lines that express a transgene introduced on a retroviral vector. For modified, transgenic animals, the lentivirus group must be used because gammaretrovectors are silenced during embryonic development. RV vectors can also be used in the delivery of toxic genes to cancer cells, which are actively dividing. Another area of application is gene discovery. The integration of the viral genome can reveal function by insertional inactivation of a gene in the

antibodies against the viral particles.

*3.2.3. Retroviral vectors (RV)*

vectors.

host cell chromosome (Somia, 2003).

 It has evolved to persist in a lifelong nonintegrated latent state without causing disease in the immune-competent host. Among the herpes family Herpes Simplex Virus type 1 (HSV-1) is an attractive vehicle because in natural infection, the virus establishes latency in neurons, a state in which viral genomes may persist for the life of the host as intranuclear episomal elements. Although the wild-type virus may be reactivated from latency under the influence of a variety of stresses, completely replication-defective and non-lytic viruses can be design (Goins et al., 2003). HSV-1 has a broad host range and does not require cell division for infection and gene expression. Accordingly, HSV may be generally useful for gene transfer to a variety of normal and disease tissues. The overall size of the HSV-1 genome (152 kb) represents an attractive feature for employing the vector for the transfer of large amounts of exogenous genetic sequences. Approximately onehalf of the HSV-1 coding sequences are nonessential for virus replication in cell culture. At least 44 kb of HSV sequence can potentially be removed in order to accommodate a transgene (Wolfe et al., 2009).

The obstacles that need to be addressed in order to take advantage of the full potential of these vectors include elimination of residual vector toxicity, design of promoter cassettes that provide sufficient level and duration of transgene expression, and targeting of transgene expression to specific cell populations through the use of tissue-specific promoters, or by altering the virus host range through modifying receptor utilization for attachment and entry (Wolfe et al., 2009).

#### *3.2.5. Sendai Virus Vectors (SeV)*

Since its isolation in 1953 in Japan, Sendai virus (SeV) has been widely used as a research tool in cell biology and in the industry, but the application of SeV as a recombinant viral vector has been investigated only recently. Sendai virus (SeV) is a nonsegmented negative-strand RNA virus belonging to the *Paramyxoviridae* family. As SeV can infect various animal cells with an exceptionally broad host range and is not pathogenic to humans, various applications have been explored for SeV as a recombinant viral vector capable of transient but strong gene expression (Nakanishi and Otsu, 2012). Its RNA nature is advantageous for applications in which chromosomal integration of exogenous genes can be undesirable. These viral vectors are currently being tested in regenerative medicine to reprogram cell genomes to a pluripotent state with a surprisingly high efficiency (Nishimura et al., 2011; MacArthur et al., 2012) and as recombinant viral vaccines for influenza prevention (Le et al., 2011).

#### **3.3. Recombinant adenoviral vectors**

Although the pathologies associated with wild-type Adenovirus (AdV) infections are gener‐ ally mild, there is a potential risk of using fully replication-competent AdV for gene transfer because the inflammatory host responses may alter organ function. There is also the possibility of overwhelming infection if AdV replication is allowed to progress when there are deficiencies in the host defense system. These situations became then a major reason to develop recombi‐ nant, non-pathogenic viral particles.

Regardless experimentation with different viral gene delivery systems, adenoviral vectors continue to be widely used for gene transfer strategies (Kovesdi et al., 1997; De Gruijl et al, 2012; Youngjoo et al, 2013; Fishbein I et al, 2013). They were also the first viral vector system to be developed. However this was more by chance than intentionally while working in the production of live adenoviral vaccines propagated in monkey cell lines. Infection of tissue culture cells with AdV vaccine accidentally contaminated with simian virus 40 (SV40) resulted in the production of the SV40 T antigen, even after removal of the SV40 virions from the AdV stocks by immunodepletion. Analysis of this adenovirus revealed that the T-antigen gene from SV40 had recombined into the E3 region of the Ad genome. This demonstrated the possibility that AdV could carry foreign genes and express them as well as demonstrating the dispensa‐ bility of the E3 genes for *in vitro* replication (Roy-Chowdhury and Horwitz, 2002; Campos et al., 2007).

The first wild type adenoviruses subjected to "vectoring" process were AdVs derived from the human serotypes 5 (Ad5) and 2 (Ad2). First-generation replication-deficient Ad5 vectors were developed by deleting the E1 genes, necessary for expression of E2 and late genes required for AdV DNA synthesis, capsid protein expression, and viral replication. Further deletions included the E3 genes which are involved in the evasion of host immune defenses but dispensable for replication of the virus *in vitro*. Therefore, because of this experimental manipulation leading to viral replication impairment, there was the need to develop a biological system capable of providing the genes required for the virus propagation. This led to the creation of the HEK 293 cell line, which was transfected with sheared adenovirus-type 5 (Ad5) genomic DNA and stably expresses the E1 genes (Graham et al., 1977).

One of the most spectacular results in gene therapy using HD-AdV was published by Kim et al., 2001. They used a gutless AdV harboring the apoE gene to treat apoE-deficient C57BL/6 mice which display spontaneous hypercholesterolemia. These mice received a single intrave‐ nous (i.v) injection of HD-AdV-ApoE at the 12th week of age. This isolated intervention managed to normalized plasma apoE concentrations and therefore diminish the plasma cholesterol level to values found in wild-type mice. However, the most outstanding finding involves the duration of the protective effect of this gene therapy, being of at least 2.5 years,

Many of these AdV vectors produced in accordance with regulated quality standards, are now being used for human clinical trials. For detailed information the reader is referred to the

The skeletal muscle gene transfer approach using Adenoviral Vectors has created controversy. There are several studies with heterogeneous efficiency rates and, in some cases, divergent outcomes. On this section we will intend to present and discuss these results as well as to introduce a novel technology that might overcome the difficulties experienced in the trans‐

following reviews: Russell, 2000; Józkowicz and Dulak, 2005; Campos, 2007.

**3.4. Adenoviral vectors in tissue-specific gene transfer: The skeletal muscle**

the lifespan of these mice.

**Ad vector generation**

second E2 (complete

third most of the

or partial)

genome. ITR and packaging seq. retained ("gutless", HC or HD-Ad)

**AdV genome deletions**

**foreign DNA insertion capacity**

**Table 1.** Comparison of adenoviral vectors generations (Russell, 2000).

**propagation in cell lines**

8kb 293-C2 (express E2) AE1-2a

35-37 kb 293 cells expressing Cre;

require helper virus

first E1 and/or E3 6.5 kb HEK293 (express E1) transient significant Possibility of the

**type of transgene expression**

transient important;

especially after repeated administration

long-lasting low or none No production of

**host immune response**

**risks**

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production of replicationcompetent adenovirus (RCA)

No production of

RCA

RCA

duction of this tissue.

Although the first-generation AdV vectors are generally considered replication defective, there is some low level expression of viral antigens that limits the duration of transgene expression *in vivo*, due to elimination of transduced cells by the cellular immune system (McConnell and Imperiale, 2004). To avoid this response and allow long-term episomal expression a new recombinant vector was developed. They are referred as "gutless" or high-capacity Adenovi‐ rus (HC-AdV) lack all viral coding sequences except the *cis*-elements required for the genome replication and encapsidation. Therefore, they need to be assisted by Helper Adenoviruses that provide all necessary replication genes in *trans*; therefore, they are also called Helper-Dependent adeno vectors (HD-AdV) (Amalfitano, 1999). In addition, these vectors have a much higher packaging capacity of ~35 kb of foreign DNA, enabling the expression of large transgenes or the inclusion of human genomic regulatory elements (Palmer and Ng, 2005). See Table 1 for comparison of the profile of different vector generations.

A variety of applications can be found for these vectors. The most widely explored are Gene Therapy for prostate, colon, cervix, ovary and CNS tumors, for genetic diseases such as Hemophilia, Duchene Muscular Dystrophy, Familial Hypercholesterolemia, Autoimmune Diabetes; viral vaccines; supplementary therapy for degenerative conditions as Parkinson, Alzheimer and Rheumatic Arthritis and production of recombinant proteins among others.

**Table 1.** Comparison of adenoviral vectors generations (Russell, 2000).

in the host defense system. These situations became then a major reason to develop recombi‐

Regardless experimentation with different viral gene delivery systems, adenoviral vectors continue to be widely used for gene transfer strategies (Kovesdi et al., 1997; De Gruijl et al, 2012; Youngjoo et al, 2013; Fishbein I et al, 2013). They were also the first viral vector system to be developed. However this was more by chance than intentionally while working in the production of live adenoviral vaccines propagated in monkey cell lines. Infection of tissue culture cells with AdV vaccine accidentally contaminated with simian virus 40 (SV40) resulted in the production of the SV40 T antigen, even after removal of the SV40 virions from the AdV stocks by immunodepletion. Analysis of this adenovirus revealed that the T-antigen gene from SV40 had recombined into the E3 region of the Ad genome. This demonstrated the possibility that AdV could carry foreign genes and express them as well as demonstrating the dispensa‐ bility of the E3 genes for *in vitro* replication (Roy-Chowdhury and Horwitz, 2002; Campos et

The first wild type adenoviruses subjected to "vectoring" process were AdVs derived from the human serotypes 5 (Ad5) and 2 (Ad2). First-generation replication-deficient Ad5 vectors were developed by deleting the E1 genes, necessary for expression of E2 and late genes required for AdV DNA synthesis, capsid protein expression, and viral replication. Further deletions included the E3 genes which are involved in the evasion of host immune defenses but dispensable for replication of the virus *in vitro*. Therefore, because of this experimental manipulation leading to viral replication impairment, there was the need to develop a biological system capable of providing the genes required for the virus propagation. This led to the creation of the HEK 293 cell line, which was transfected with sheared adenovirus-type

Although the first-generation AdV vectors are generally considered replication defective, there is some low level expression of viral antigens that limits the duration of transgene expression *in vivo*, due to elimination of transduced cells by the cellular immune system (McConnell and Imperiale, 2004). To avoid this response and allow long-term episomal expression a new recombinant vector was developed. They are referred as "gutless" or high-capacity Adenovi‐ rus (HC-AdV) lack all viral coding sequences except the *cis*-elements required for the genome replication and encapsidation. Therefore, they need to be assisted by Helper Adenoviruses that provide all necessary replication genes in *trans*; therefore, they are also called Helper-Dependent adeno vectors (HD-AdV) (Amalfitano, 1999). In addition, these vectors have a much higher packaging capacity of ~35 kb of foreign DNA, enabling the expression of large transgenes or the inclusion of human genomic regulatory elements (Palmer and Ng, 2005). See

A variety of applications can be found for these vectors. The most widely explored are Gene Therapy for prostate, colon, cervix, ovary and CNS tumors, for genetic diseases such as Hemophilia, Duchene Muscular Dystrophy, Familial Hypercholesterolemia, Autoimmune Diabetes; viral vaccines; supplementary therapy for degenerative conditions as Parkinson, Alzheimer and Rheumatic Arthritis and production of recombinant proteins among others.

5 (Ad5) genomic DNA and stably expresses the E1 genes (Graham et al., 1977).

Table 1 for comparison of the profile of different vector generations.

nant, non-pathogenic viral particles.

176 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

al., 2007).

One of the most spectacular results in gene therapy using HD-AdV was published by Kim et al., 2001. They used a gutless AdV harboring the apoE gene to treat apoE-deficient C57BL/6 mice which display spontaneous hypercholesterolemia. These mice received a single intrave‐ nous (i.v) injection of HD-AdV-ApoE at the 12th week of age. This isolated intervention managed to normalized plasma apoE concentrations and therefore diminish the plasma cholesterol level to values found in wild-type mice. However, the most outstanding finding involves the duration of the protective effect of this gene therapy, being of at least 2.5 years, the lifespan of these mice.

Many of these AdV vectors produced in accordance with regulated quality standards, are now being used for human clinical trials. For detailed information the reader is referred to the following reviews: Russell, 2000; Józkowicz and Dulak, 2005; Campos, 2007.

#### **3.4. Adenoviral vectors in tissue-specific gene transfer: The skeletal muscle**

The skeletal muscle gene transfer approach using Adenoviral Vectors has created controversy. There are several studies with heterogeneous efficiency rates and, in some cases, divergent outcomes. On this section we will intend to present and discuss these results as well as to introduce a novel technology that might overcome the difficulties experienced in the trans‐ duction of this tissue.

Direct gene transfer into skeletal muscle cells *in vitro* and *in vivo* using either plasmid DNA or recombinant viruses has medical applications in vaccination and gene therapy and also has been widely used in studies of developmental and physiological regulation of muscle gene expression (Hallauer et al., 2000). Many factors regarding muscle tissue barriers, immune response, systemic dissemination, potential toxicity and specific properties of each viral system need to be taken into account when selecting the proper approach for skeletal muscle. It is well known that skeletal muscle is a highly developed and organized tissue in which the constituent myofibers become post-mitotic in fetal life. The mononucleated myogenic precursor cells (satellite cells) that are located between the extracellular matrix and the plasma membrane of myofibers are known to be capable of fusing together or with preexisting myofibers in response to various types of stimuli, mainly to injury (Chargé et al., 2004). These satellite cells are relatively easy to isolate and cultivate *in vitro* and can also be efficiently transduced using virtually any viral vector in contrast with the mature multinucleated myofibers. Thus, is believed that viral transduction during skeletal muscle maturation might require mitotically active myoblasts (van Deutekom et al., 1998). However, because some of the viral vectors have been shown to transduce post-mitotic, immature myofibers *in vitro* and *in vivo* other factors are also likely to be involved in the poor level of viral transduction of mature myofibers (Wolff et al., 1990; Acsadi et al., 1994 and Huard et al., 1996;).

Despite this wide range of viral delivery systems, *Adenoviral (AdV) vectors* are probably the most prominent ones in this matter and have been extensible use to deliver genes into skeletal muscle. However, several obstacles have been identified in the application of adenovirus as gene delivery vehicles to skeletal muscle (Acsadi et al., 1994 and van Deutekom et al., 1998). The major limitations facing first generation adenoviral gene transfer to skeletal muscle are (1) the lack of transgene persistence due to the immune rejection of transduced myofibers; (2) the relatively low insert carrying capacity; (3) the reduced viral transducibility during muscle maturation; and (4) repeated administration associated with the production of neutralizing antibodies is limited to the viral capsid (Cao et al., 2001). During experiments of AdV gene transfer in animals of different ages it became clear that the transduction efficiency was related with the maturation state of the muscle. While the skeletal muscle of newborn mice achieved high levels of AdV infection, the mature muscle from adult animals was significantly less susceptible to infection under the same conditions (Huard et al., 1995). Here the high adeno‐ viral transduction of newborn myofibers could be explained due partly to transduction of myoblasts and partly to the higher levels of CAR in these myofibers (Nalbantoglu et al., 1999). Several studies have shown that in developing human, mouse and rat muscle, expres‐ sion of the primary AdV membrane receptor CAR is severely downregulated even at early ages with CAR mRNA being barely detectable in adult myofibers (Nalbantoglu et al., 1999). Furthermore, it has been demonstrated that forced expression of CAR in myotubes by different approaches, such as RAdV encoding hCAR or transgenic mice overexpressing the receptor, overcomes the poor AdV mediated transducibility of these cells (Nalbantoglu et al., 2001 and Kimura et al., 2001). On the other hand, basal lamina and glycocalyx surrounding mature skeletal muscle cells appear to be an anatomical barrier that may limit the access and distri‐ bution of exogenously introduced virus. (van Deutekom et al., 1998 and Cao et al., 2001). It has been reported that the extracellular matrix of mature myofibers may form a physical barrier and prevent the passage of some viral particles that are too big to pass through its pores, which are estimated at 40 nm in size. Adenoviral particles are about 70 nm and 100 nm in diameter and appear to be too large to penetrate the pores of the basal lamina (Cao et al., 2001). Re‐ gardless these difficulties some authors have published high rates of skeletal muscle trans‐ duction using AdV. In 2002, Sapru et al., was able to achieve nearly 100% of transduced fibers in the adult rat Soleus and more than 80% in the Tibialis Anterior muscle when infected with an adenoviral vector harbouring the cDNA of the GFP under the control of the CMV promoter (AdVCMV-GFP). These authors claim that the viral titer used was an important factor since they could increase the number of transduced fiber when the viral dose was doubled. Other major factor regarding skeletal muscle infection with AdV and AAV seems to be the fiber composition of the muscle, with suggested preferential transduction of slow fibers (Pruchnic et al., 2000 and Sapru et al., 2002). New AdV vectors lacking all viral genes, the *Helper-Dependent AdV vectors*, show a markedly decreased immunogenicity and hence, an improved persistence of transgene expression in muscle *in vivo* (Bilbao et al., 2005). These observations suggest that the limitations regarding the immunogenicity with the use of adenoviral vectors are being overcome. However, the inability of adenoviral vectors to efficiently transduce mature myofibers remains a major hurdle facing the widespread application of adenoviral gene

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transfer to skeletal muscle (van Deutekom et al., 1998).

*Retroviral vectors (RV)* can infect dividing myoblasts with a high efficiency although they remain incapable of infecting post mitotic myotubes or myofibers (Miller et al., 1990 and Salvatori et al., 1993). In addition, the ability to become stably integrated into the host cell genome, which can provide long-term, stable expression of the delivered gene, may also represent a risk for insertional mutagenesis. As mentioned in Section 3.2 of the present chapter, other limitations to the use of retroviruses are the gene insert capacity (less than 7 kb) and the relatively low production titers (105 –106 plaque-forming units per milliliter (pfu/mL)). The recombinant vectors obtained from the *Herpes Simplex Virus type 1 (HSV-1)* can persist in the host cell in a nonintegrated state and be prepared at adequately high titers (107 –109 pfu / ml). They are capable of transducing muscle cells in most maturation stages while carrying large DNA fragments. However, are still unable to highly penetrate and transduce mature myofibers (Huard et al., 1997). *Adeno-associated derived viral vectors (AAV)* have also been used to approach muscle cells. Although a long-term gene expression (up to 18 months) and a high efficiency of mature myofibers transduction have been observed in mouse skeletal muscle, the application of adeno-associated viral vectors for gene therapy may be limited by their restrictive gene insert capacity (Pruchnic et al., 2000). In the last few years a novel gene transfer viral vector has been used into skeletal muscle, the *recombinant Sendai virus (SeV) vectors*. As explained before, the wild SeV is a non-segmented negative-strand RNA virus belonging to the *Para‐ myxoviridae* family that can infect various animal cells with an exceptionally broad host range. Shiotani et al., 2001 accomplished a significant overexpression of hIGF-1 in the adult rat Tibialis Anterior muscle when injected the tissue with a recombinant SeV vector (hIGF-1/SeV).

Despite this wide range of viral delivery systems, *Adenoviral (AdV) vectors* are probably the most prominent ones in this matter and have been extensible use to deliver genes into skeletal muscle. However, several obstacles have been identified in the application of adenovirus as gene delivery vehicles to skeletal muscle (Acsadi et al., 1994 and van Deutekom et al., 1998). The major limitations facing first generation adenoviral gene transfer to skeletal muscle are (1) the lack of transgene persistence due to the immune rejection of transduced myofibers; (2) the relatively low insert carrying capacity; (3) the reduced viral transducibility during muscle maturation; and (4) repeated administration associated with the production of neutralizing antibodies is limited to the viral capsid (Cao et al., 2001). During experiments of AdV gene transfer in animals of different ages it became clear that the transduction efficiency was related with the maturation state of the muscle. While the skeletal muscle of newborn mice achieved high levels of AdV infection, the mature muscle from adult animals was significantly less susceptible to infection under the same conditions (Huard et al., 1995). Here the high adeno‐ viral transduction of newborn myofibers could be explained due partly to transduction of myoblasts and partly to the higher levels of CAR in these myofibers (Nalbantoglu et al., 1999). Several studies have shown that in developing human, mouse and rat muscle, expres‐ sion of the primary AdV membrane receptor CAR is severely downregulated even at early ages with CAR mRNA being barely detectable in adult myofibers (Nalbantoglu et al., 1999). Furthermore, it has been demonstrated that forced expression of CAR in myotubes by different approaches, such as RAdV encoding hCAR or transgenic mice overexpressing the receptor, overcomes the poor AdV mediated transducibility of these cells (Nalbantoglu et al., 2001 and Kimura et al., 2001). On the other hand, basal lamina and glycocalyx surrounding mature skeletal muscle cells appear to be an anatomical barrier that may limit the access and distri‐ bution of exogenously introduced virus. (van Deutekom et al., 1998 and Cao et al., 2001). It has been reported that the extracellular matrix of mature myofibers may form a physical barrier and prevent the passage of some viral particles that are too big to pass through its pores, which are estimated at 40 nm in size. Adenoviral particles are about 70 nm and 100 nm in diameter and appear to be too large to penetrate the pores of the basal lamina (Cao et al., 2001). Re‐ gardless these difficulties some authors have published high rates of skeletal muscle trans‐ duction using AdV. In 2002, Sapru et al., was able to achieve nearly 100% of transduced fibers in the adult rat Soleus and more than 80% in the Tibialis Anterior muscle when infected with an adenoviral vector harbouring the cDNA of the GFP under the control of the CMV promoter (AdVCMV-GFP). These authors claim that the viral titer used was an important factor since they could increase the number of transduced fiber when the viral dose was doubled. Other major factor regarding skeletal muscle infection with AdV and AAV seems to be the fiber composition of the muscle, with suggested preferential transduction of slow fibers (Pruchnic et al., 2000 and Sapru et al., 2002). New AdV vectors lacking all viral genes, the *Helper-Dependent AdV vectors*, show a markedly decreased immunogenicity and hence, an improved persistence of transgene expression in muscle *in vivo* (Bilbao et al., 2005). These observations suggest that the limitations regarding the immunogenicity with the use of adenoviral vectors are being overcome. However, the inability of adenoviral vectors to efficiently transduce mature myofibers remains a major hurdle facing the widespread application of adenoviral gene transfer to skeletal muscle (van Deutekom et al., 1998).

Direct gene transfer into skeletal muscle cells *in vitro* and *in vivo* using either plasmid DNA or recombinant viruses has medical applications in vaccination and gene therapy and also has been widely used in studies of developmental and physiological regulation of muscle gene expression (Hallauer et al., 2000). Many factors regarding muscle tissue barriers, immune response, systemic dissemination, potential toxicity and specific properties of each viral system need to be taken into account when selecting the proper approach for skeletal muscle. It is well known that skeletal muscle is a highly developed and organized tissue in which the constituent myofibers become post-mitotic in fetal life. The mononucleated myogenic precursor cells (satellite cells) that are located between the extracellular matrix and the plasma membrane of myofibers are known to be capable of fusing together or with preexisting myofibers in response to various types of stimuli, mainly to injury (Chargé et al., 2004). These satellite cells are relatively easy to isolate and cultivate *in vitro* and can also be efficiently transduced using virtually any viral vector in contrast with the mature multinucleated myofibers. Thus, is believed that viral transduction during skeletal muscle maturation might require mitotically active myoblasts (van Deutekom et al., 1998). However, because some of the viral vectors have been shown to transduce post-mitotic, immature myofibers *in vitro* and *in vivo* other factors are also likely to be involved in the poor level of viral transduction of mature myofibers (Wolff

178 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

*Retroviral vectors (RV)* can infect dividing myoblasts with a high efficiency although they remain incapable of infecting post mitotic myotubes or myofibers (Miller et al., 1990 and Salvatori et al., 1993). In addition, the ability to become stably integrated into the host cell genome, which can provide long-term, stable expression of the delivered gene, may also represent a risk for insertional mutagenesis. As mentioned in Section 3.2 of the present chapter, other limitations to the use of retroviruses are the gene insert capacity (less than 7 kb) and the

recombinant vectors obtained from the *Herpes Simplex Virus type 1 (HSV-1)* can persist in the

They are capable of transducing muscle cells in most maturation stages while carrying large DNA fragments. However, are still unable to highly penetrate and transduce mature myofibers (Huard et al., 1997). *Adeno-associated derived viral vectors (AAV)* have also been used to approach muscle cells. Although a long-term gene expression (up to 18 months) and a high efficiency of mature myofibers transduction have been observed in mouse skeletal muscle, the application of adeno-associated viral vectors for gene therapy may be limited by their restrictive gene insert capacity (Pruchnic et al., 2000). In the last few years a novel gene transfer viral vector has been used into skeletal muscle, the *recombinant Sendai virus (SeV) vectors*. As explained before, the wild SeV is a non-segmented negative-strand RNA virus belonging to the *Para‐ myxoviridae* family that can infect various animal cells with an exceptionally broad host range. Shiotani et al., 2001 accomplished a significant overexpression of hIGF-1 in the adult rat Tibialis Anterior muscle when injected the tissue with a recombinant SeV vector (hIGF-1/SeV). They indicate a favorable gene delivery to mitotic myoblasts, post-mitotic immature and mature

plaque-forming units per milliliter (pfu/mL)). The

–109

pfu / ml).

–106

host cell in a nonintegrated state and be prepared at adequately high titers (107

et al., 1990; Acsadi et al., 1994 and Huard et al., 1996;).

relatively low production titers (105

myofibers.

On the next section we will introduce the novel combination of Magnetic Nanoparticles and Recombinant Adenoviral Vectors as an efficient alternative for gene delivery in transductionresistant differentiated skeletal muscle cells.

(or zeta-) potential of all types of viral particles in aqueous media allow their assembling with cationic species and particles due to electrostatically induced aggregation (Scherer et al., 2002). The resulting magnetic-viral vectors complexes are then forced into sedimentation over

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As detailed in Section 2.3, one of the most remarkable aspects of magnetofection enhancing viral gene transfer is the lack of need for virus interaction with cellular receptors. This has a particular significance for adenoviral vectors. It is known that for all the Wild-Type (except Subgroup B) and Recombinant Adenovirus cell entry takes place through receptor-mediated endocytosis via the Coxsackie–Adenovirus Receptor (CAR) (Meier and Greber, 2003). Various cell types differ widely in their level of CAR expression, and this may be a limiting factor for the transduction efficiency achievable with Adenovirus (Chorny et al., 2006). When AdVs are complex with MNPs all the viral surface proteins become hidden and unreachable for the CAR receptor. The cell entry in then achieved by unspecific clathrin-mediated endocytosis (Plank

In order to find the optimal magnetic vector formulations for plasmid, siRNA and viral vector delivery, Plank and co-workers have aimed at maximal association with the magnetic com‐ ponent but avoiding an excess of magnetic particles (Plank et al., 2011). They have published that an excess of magnetic nanoparticles can inhibit transfection/transduction efficiency and cause toxicity (Tresilwised et al., 2010 and Sanchez-Antequera et al., 2011). Therefore, finding the optimal MNPs-to-nucleic acid ratios (about 0.5–1 units of iron weight per unit of the nucleic acid weight for triplexes with an enhancer) as well as MNPs-to-virus ratios (2.5–10 fg iron per virus particle further referred to as fg Fe/VP) have turned out useful for a variety of magnetic nanoparticle types (Sanchez-Antequera et al., 2011). The complexes formulated in this way were efficient and hardly toxic in delivery of DNA and siRNA as well in delivery of adenoviral and lentiviral vectors *in vitro* and *ex vivo*. Particularly to this regard there are several studies that have demonstrated the higher efficiency of this method when compare with traditional viral transduction using Adenovirus, Adeno-associated Virus, Baculovirus, Lentivirus and Retrovirus (Chan et al., 2005; Chorny et al., 2009; Hughes et al., 2001; Kaikkonen et al., 2008; Mah et al., 2000 and 2002; Morizono et al., 2009; Raty et al., 2004; Tresilwised et al., 2010 and

Regarding our experimental field, development of reliable techniques for manipulation of gene expression in mature skeletal muscle fibers is critical for understanding molecular mechanisms involved in both physiology and physiopathology. As explained before, differentiated skeletal muscle myotubes and myofibers are refractory to most standard protocols for gene transfer in vitro and in vivo and the use of adenoviral vectors offers relatively low efficiency. It is believed that a maturation-dependent loss of the CAR receptor together with structural and biochemical changes are responsible for these decreased transduction efficiencies (Nalbantoglu et al., 1999). It has been proposed that these limitations can be overcome by achieving adenoviral cellular uptake via a CAR independent pathway using genetic modifications of the capsid proteins or chemical modifications of the virus. However, these strategies are not sufficient for rapid infection of the cells at the target site, as the delivery process itself is diffusion-limited (Haim et al., 2005 and Schillinger et al., 2005). Here, magnetofection provided us a powerful,

the cell monolayer when a magnetic field is applied.

et al., 2011).

2012).

#### **3.5. Use of magnetic nanoparticles and magnetic fields to enhance viral vector-based gene delivery**

Nowadays, the novel association of non-viral or viral vector-based gene delivery with nanotechnology offers the possibility to develop more efficient gene transfer strategies for a number of applications. In 2002 the concept of Magnetofection was first published by Scherer et al. Here, the Magnetic Drug Targeting (MDT) approach (Widder et al., 1978) classically used to concentrate magnetically responsive therapeutic complexes in target areas of the body by means of external gradient magnetic fields was applied for gene delivery. Therefore, Magne‐ tofection is based on the association of Magnetic Nanoparticles (MNPs) with non-viral or viral vectors in order to optimize gene delivery when exposed to a magnetic field (Scherer et al., 2002).

There are currently several synthetic formulations of MNPs commercially available for biomedical applications such as cell separation, drug/gene delivery, magnetic resonance imaging (MRI) and hyperthermia (Gupta et al., 2005). Despite the differences, they all need to comprise some basic functionality to allow them to be associated with a gene delivery vector. Furthermore, the magnetic properties of these particles have to be sufficient to concentrate the vector at the target cells under a magnetic force and the formulation has to be biocompatible enough for application in living cells or organisms (Plank et al., 2011). Their general structure is based on a magnetic core of magnetite (Fe3O4) or maghemite (g-Fe2O3) coated with synthetic polymers that provides both protection and biological functionality. Occasionally specific organic linkers are added to this structure to generate new attachment sites for drugs or gene vectors (Yallapu et al., 2010). (Figure 5).

**Figure 5.** General Structure of a Magnetic Nanoparticle

For association with nucleic acids and/or viral particles, coatings comprised of cationic polymers such as polyethylenimine (PEI) are particularly useful (Mykhaylyk et al., 2007). The negatively charged phosphate backbone of nucleic acids as well as the negative electrokinetic (or zeta-) potential of all types of viral particles in aqueous media allow their assembling with cationic species and particles due to electrostatically induced aggregation (Scherer et al., 2002). The resulting magnetic-viral vectors complexes are then forced into sedimentation over the cell monolayer when a magnetic field is applied.

On the next section we will introduce the novel combination of Magnetic Nanoparticles and Recombinant Adenoviral Vectors as an efficient alternative for gene delivery in transduction-

**3.5. Use of magnetic nanoparticles and magnetic fields to enhance viral vector-based gene**

Nowadays, the novel association of non-viral or viral vector-based gene delivery with nanotechnology offers the possibility to develop more efficient gene transfer strategies for a number of applications. In 2002 the concept of Magnetofection was first published by Scherer et al. Here, the Magnetic Drug Targeting (MDT) approach (Widder et al., 1978) classically used to concentrate magnetically responsive therapeutic complexes in target areas of the body by means of external gradient magnetic fields was applied for gene delivery. Therefore, Magne‐ tofection is based on the association of Magnetic Nanoparticles (MNPs) with non-viral or viral vectors in order to optimize gene delivery when exposed to a magnetic field (Scherer et al.,

There are currently several synthetic formulations of MNPs commercially available for biomedical applications such as cell separation, drug/gene delivery, magnetic resonance imaging (MRI) and hyperthermia (Gupta et al., 2005). Despite the differences, they all need to comprise some basic functionality to allow them to be associated with a gene delivery vector. Furthermore, the magnetic properties of these particles have to be sufficient to concentrate the vector at the target cells under a magnetic force and the formulation has to be biocompatible enough for application in living cells or organisms (Plank et al., 2011). Their general structure is based on a magnetic core of magnetite (Fe3O4) or maghemite (g-Fe2O3) coated with synthetic polymers that provides both protection and biological functionality. Occasionally specific organic linkers are added to this structure to generate new attachment sites for drugs

resistant differentiated skeletal muscle cells.

180 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

or gene vectors (Yallapu et al., 2010). (Figure 5).

Magnetic Core

Polymer Coating

**Figure 5.** General Structure of a Magnetic Nanoparticle

Organic Linker

For association with nucleic acids and/or viral particles, coatings comprised of cationic polymers such as polyethylenimine (PEI) are particularly useful (Mykhaylyk et al., 2007). The negatively charged phosphate backbone of nucleic acids as well as the negative electrokinetic

Drug/Gene Vector

**delivery**

2002).

As detailed in Section 2.3, one of the most remarkable aspects of magnetofection enhancing viral gene transfer is the lack of need for virus interaction with cellular receptors. This has a particular significance for adenoviral vectors. It is known that for all the Wild-Type (except Subgroup B) and Recombinant Adenovirus cell entry takes place through receptor-mediated endocytosis via the Coxsackie–Adenovirus Receptor (CAR) (Meier and Greber, 2003). Various cell types differ widely in their level of CAR expression, and this may be a limiting factor for the transduction efficiency achievable with Adenovirus (Chorny et al., 2006). When AdVs are complex with MNPs all the viral surface proteins become hidden and unreachable for the CAR receptor. The cell entry in then achieved by unspecific clathrin-mediated endocytosis (Plank et al., 2011).

In order to find the optimal magnetic vector formulations for plasmid, siRNA and viral vector delivery, Plank and co-workers have aimed at maximal association with the magnetic com‐ ponent but avoiding an excess of magnetic particles (Plank et al., 2011). They have published that an excess of magnetic nanoparticles can inhibit transfection/transduction efficiency and cause toxicity (Tresilwised et al., 2010 and Sanchez-Antequera et al., 2011). Therefore, finding the optimal MNPs-to-nucleic acid ratios (about 0.5–1 units of iron weight per unit of the nucleic acid weight for triplexes with an enhancer) as well as MNPs-to-virus ratios (2.5–10 fg iron per virus particle further referred to as fg Fe/VP) have turned out useful for a variety of magnetic nanoparticle types (Sanchez-Antequera et al., 2011). The complexes formulated in this way were efficient and hardly toxic in delivery of DNA and siRNA as well in delivery of adenoviral and lentiviral vectors *in vitro* and *ex vivo*. Particularly to this regard there are several studies that have demonstrated the higher efficiency of this method when compare with traditional viral transduction using Adenovirus, Adeno-associated Virus, Baculovirus, Lentivirus and Retrovirus (Chan et al., 2005; Chorny et al., 2009; Hughes et al., 2001; Kaikkonen et al., 2008; Mah et al., 2000 and 2002; Morizono et al., 2009; Raty et al., 2004; Tresilwised et al., 2010 and 2012).

Regarding our experimental field, development of reliable techniques for manipulation of gene expression in mature skeletal muscle fibers is critical for understanding molecular mechanisms involved in both physiology and physiopathology. As explained before, differentiated skeletal muscle myotubes and myofibers are refractory to most standard protocols for gene transfer in vitro and in vivo and the use of adenoviral vectors offers relatively low efficiency. It is believed that a maturation-dependent loss of the CAR receptor together with structural and biochemical changes are responsible for these decreased transduction efficiencies (Nalbantoglu et al., 1999). It has been proposed that these limitations can be overcome by achieving adenoviral cellular uptake via a CAR independent pathway using genetic modifications of the capsid proteins or chemical modifications of the virus. However, these strategies are not sufficient for rapid infection of the cells at the target site, as the delivery process itself is diffusion-limited (Haim et al., 2005 and Schillinger et al., 2005). Here, magnetofection provided us a powerful, accessible and efficient tool for transducing differentiated myotubes of the C2C12 cell line (Pereyra A et al., 2011-Posters Sessions). A first generation (E1/E3-deleted), serotype 5, Recombinant Adenoviral vector harboring de cDNA of the Green Fluorescent Protein (RAdV-GFP) under the control of the CMV promoter was constructed in our laboratory. This vector was incubated with Atto550PEI-Mag2, a magnetic nanoparticle conjugated with a red fluo‐ rescent dye that allows particle tracking during the cellular uptake and internalization. Then the [RAdV-GFP-Atto550PEI-Mag2] complexes were incorporated to the supernatant of the mature myotubes cultures. The magnetic field required for sedimentation was provided by a commercial plate (Oz Biosciences®, Marseille, France) composed of cylindrical-permanent-Nd-Fe-B magnets. The same protocol was tested in undifferentiated C2C12 myoblast cultures and conventional RAdV transduction experiments using the same viral multiplicity of infection (MOI) were also performed for efficiency comparisons. (Figure 6)

**Figure 6.** Magnetofection in cell culture. This basic scheme shows the process of magnetofection in cultured cells. The pre-incubated RAdV-MNPs complexes are introduced to the cell culture. Then the culture plate is exposed to a mag‐ netic field created by the magnetic plate placed under the cells.

As showed in Figure 7, the poor RadV-GFP transduction of mature myotubes was overcome by magnetofection. In myoblasts, were the conventional transduction protocols show an acceptable efficiency, the magnetofection method displayed an enhancer effect. The intracel‐ lular localization of the magnetic nanoparticles can be seen in Figure 8.

1

1

**Myoblasts+RAd-GFP+Atto550- PEI-Mag2 with exposure to magnetic field**

Gene Delivery Systems

183

http://dx.doi.org/10.5772/56869

**Myoblasts+RAd-GFP MOI 60**

2011-Posters Sessions).

**Myoblasts+RAd-GFP+Atto550- PEI-Mag2 without exposure to magnetic field**

**Figure 7.** Magnetofection in mature C2C12 myotubes and myoblasts. The conventional RAdV transduction system was compared against magnetofection. The images were obtained 48 hs after incubation. The green fluorescence corresponds to the expression of the GFP protein encoded by the viral genome. Magnification 40X (Pereyra A et al.,

**Figure 8.** This picture was taken with a Double Band filter in order to appreciate the cellular co-localization of the green and red (Atto550-PEI-Mag2) fluorescence. Magnification 40X (Pereyra A et al., 2011-Posters Sessions).

1

accessible and efficient tool for transducing differentiated myotubes of the C2C12 cell line (Pereyra A et al., 2011-Posters Sessions). A first generation (E1/E3-deleted), serotype 5, Recombinant Adenoviral vector harboring de cDNA of the Green Fluorescent Protein (RAdV-GFP) under the control of the CMV promoter was constructed in our laboratory. This vector was incubated with Atto550PEI-Mag2, a magnetic nanoparticle conjugated with a red fluo‐ rescent dye that allows particle tracking during the cellular uptake and internalization. Then the [RAdV-GFP-Atto550PEI-Mag2] complexes were incorporated to the supernatant of the mature myotubes cultures. The magnetic field required for sedimentation was provided by a commercial plate (Oz Biosciences®, Marseille, France) composed of cylindrical-permanent-Nd-Fe-B magnets. The same protocol was tested in undifferentiated C2C12 myoblast cultures and conventional RAdV transduction experiments using the same viral multiplicity of infection

**Figure 6.** Magnetofection in cell culture. This basic scheme shows the process of magnetofection in cultured cells. The pre-incubated RAdV-MNPs complexes are introduced to the cell culture. Then the culture plate is exposed to a mag‐

As showed in Figure 7, the poor RadV-GFP transduction of mature myotubes was overcome by magnetofection. In myoblasts, were the conventional transduction protocols show an acceptable efficiency, the magnetofection method displayed an enhancer effect. The intracel‐

lular localization of the magnetic nanoparticles can be seen in Figure 8.

netic field created by the magnetic plate placed under the cells.

(MOI) were also performed for efficiency comparisons. (Figure 6)

182 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

**Figure 7.** Magnetofection in mature C2C12 myotubes and myoblasts. The conventional RAdV transduction system was compared against magnetofection. The images were obtained 48 hs after incubation. The green fluorescence corresponds to the expression of the GFP protein encoded by the viral genome. Magnification 40X (Pereyra A et al., 2011-Posters Sessions).

1 **Figure 8.** This picture was taken with a Double Band filter in order to appreciate the cellular co-localization of the green and red (Atto550-PEI-Mag2) fluorescence. Magnification 40X (Pereyra A et al., 2011-Posters Sessions).


**Author details**

(14)

+5,035 ×10-4(1-2x) T (15)

For our degenerate electrons gas the Seebeck constant is described by the formula:

*α* = (*πkB*)2*T* (*s* + 1) 3*eEF*

phonons.

small in the superlattice [17].

146 Optoelectronics - Advanced Materials and Devices

The fundamental main ideas of this work are:

tice which is the key of an infrared detector.

**6. Conclusions**

where the collision time τ Es-(1/2). This permits us to estimate the Fermi energy at EF = 91 meV (in "Figure 4(a)"), with s= 2.03 corresponding to electrons diffusion by ionized impurities, in agreement with the calculated EF=88 meV in formula (13). In intrinsic regime for T>150 K, the measure of the slope of the curve RH T3/2 indicates a gap Eg = 98 meV which agree well with calculated Eg (Γ, 300 K) = E1-HH1= 105 meV. Here αT-3/2 indicates electrons scattering by

This HgTe/CdTe superlattice is a stable alternative for application in far infrared optoelec‐ tronic devices than the random alloys Hg0.99Cd0.01Te because the small composition x=0.01,

is difficult to obtain with precision while growing the ternary alloys and the transverse ef‐ fective masse in superlattice is two orders higher than in the alloy. Thus the tunnel length is

The formalism used here predicts that the system is semiconductor, for our HgTe to CdTe thickness ratio d1/d2 = 2.69, when d2 < 100 nm. In our case, d2=3.2 nm and Eg (Γ, 4.2 K) = 48 meV. In spite of it, the sample exhibits the features typical for the semiconductor n-type con‐ duction mechanism. In the used temperature range, this simple is a far-infrared detector, narrow gap and two-dimensional n-type semiconductor. Note that we had observed a semi‐

metallic conduction mechanism in the quasi 2D p type HgTe/CdTe superlattice [18].

of the superlattice; were performed in the envelope function formalism.

with Eg (Γ, 300 K) =100 meV given by the empiric formula for Hg1-xCdxTe [16]

Eg(x,T)= -0,302+1,93x-0,810x²+0,832x3

Abdelhakim Nafidi

Laboratory of Condensed Matter Physics and Nanomaterials for Renewable Energy Univer‐ sity Ibn-Zohr 80000 Agadir, Morocco


**2. Materials and Methods**

Participants were recruited from consecutive referrals by family doctors to the rehabilitative treatment programs for obesity at the inpatient unit of the Villa Garda Hospital Department of Eating and Weight Disorders during the years 2016–2019. Patients were eligible for this study if they were aged between 18 and 65 years, had a BMI <sup>≥</sup> 30.0 kg/m2, and at least one weight loss-responsive comorbidity (i.e., type 2 diabetes, cardiovascular disease, sleep apnea, severe joint disease, two or more cardiovascular risk factors), as defined by Adult Treatment Panel III [8]. The criteria for exclusion were pregnancy or lactation, medications that affect body weight, medical comorbidities associated with weight loss, severe psychiatric disorders (i.e., bulimia nervosa, acute psychotic disorders, substance use disorders), use of a walker, and the need in assistance/support with walking.

As per the Italian National Health System's National ethical guidelines, this study was classed as a routine service assessment rather than research per se, as all the procedures used for treatment and assessment were performed as routine clinical practice, and therefore no ethical clearance was necessary. That being said, each patient provided written informed consent to the collection and processing of their anonymous clinical data in the service-level research setting.

All data were collected on the second day of admission to the programs. Specifically, BMI was determined using the standard formula of body weight (kg) divided by height (m2) following measurement of body weight and height using medical weighing scales (Seca Digital Wheelchair Scale Model 664, Hamburg, Germany) and a stadiometer (Wall-Mounted Mechanical Height Rod Model 00051A; Wunder, REA (MI), Italy), respectively. The scale was calibrated for accuracy by an external accredited laboratory every two months. For the purposes of these measurements, participants were weighed in the morning (12 h after eating) wearing only lightweight clothes and no shoes and standing with minimal movement with hands by their sides. Body weight was measured once for each participant to the nearest 0.1 kg.

Physical performance was assessed by means of the six-minute walking test (6MWT) [9] according to international guidelines [10]. The 6MWT was performed along a 20 m long corridor in the department, marked with tape on the floor every 2 m; starting and finishing points were also marked on the floor in a similar fashion. Before the start and at the end of each test, pulse, respiratory rate, and oxygen saturation were measured. The patients were instructed to walk as fast as they could, but were allowed to stop or rest during the test if necessary. All participants concluded the test without breaks. The specific reference equation for predicting distance walked in six minutes in adult subjects with obesity [9] was used to assess the difference between the predicted and real 6MWT scores. The patients walking as far as or farther than predicted were included in Group H (i.e., obesity with a higher 6MWT score than expected), and the patients walking less than predicted were allocated to Group L (i.e., obesity with a lower 6MWT score than expected).

The quality of life was assessed using the validated Italian version of the Short Form-36 (SF-36)—a generic health related quality-of-life questionnaire [11,12]. The SF-36 incorporates questions about (role) functioning and satisfaction with various life domains; it consists of 36 questions, and assesses four domains related to the physical component of quality of life (Physical Functioning, Physical Role Functioning, Bodily Pain, General Health Perception), and four domains related to the mental component (Vitality, Social Functioning, Emotional Role Functioning, and Mental Health). SF-36 scale scores range from 0 to 100; a higher score indicates a better quality of life.


**4. Discussion: Main Clusters on Legaltech**

To analyze in which scientific fields or research clusters Legaltech-related works are present, a scientific community detection software, vosviewer, has been used (Van Eck and Waltman 2010). This software has proven to be useful in the analysis of many fields of knowledge such as medicine (Garrido-Cardenas et al. 2020), social sciences (Muyor-Rodriguez et al. 2019) or engineering (Salmeron-Manzano and Manzano-Agugliaro 2018). Thus, Figure 3 was obtained. This figure shows 4 different clusters that currently exist in the scientific literature, which can be distinguished according to the different colors of each one. The clusters are linked by the common keywords of all the documents analyzed. For each cluster, the main associated keywords are summarized in Table 3, and finally, a name is given to encompass the topic of each cluster.

**Figure 3.** Main clusters detected on Legaltech documents.

The first cluster is focused on computer science as verified by the main keywords involved: Algorithms, artificial intelligence, law, legal tech, legal technologies, machine learning, natural language processing. Regarding Artificial Intelligence, the first paper in this regard was "Can the mechanization of law triumph over lawyers?" from 2017 (Fortuit and Hamidou 2017), should be highlighted. The use of machine learning (ML) techniques for legal analysis and decision making in the U.S. judicial system has recently been analyzed (Delgado 2019), concluding how ML came to be adopted as a standard tool for automating fact discovery for high-stakes civil litigation.

Regarding natural language processing (NLP), there are five areas of legal activity where NLP is playing an increasing role (Dale 2019):

The second cluster is focused on Justice, as evidenced by the main keywords involved: Access to justice, design thinking, law students, rule of law. Within this topic there are works within what one could classify as philosophy of law, who argue that advances in equal access to justice and the rule of law lie primarily in the introduction of lawyers' own cognitive operations in contexts where human lawyers cannot be deployed for purely economic reasons (Gowder 2018).

The third cluster is focused on the legal profession, as can be seen from the main keywords involved: Legal profession, legal services, neo-liberalism, professional regulation. Within the field of legal services, it is worth mentioning the analysis of automated Online Dispute Resolution (ODR), where 3 cases can be identified (Barnett and Treleaven 2018):

The fourth cluster is focused on Legal design as evidenced by the main keywords involved: Blockchain, legal design, smart contract. The blockchain technology has several legal consequences and the one with the greatest need for legal regulation are cryptocurrencies such as bitcoins (Salmeron-Manzano 2017). Legal design aims to apply human-centered design to the world of law, to make legal systems and services more human-centered, usable, and satisfying (Hagan 2017). In short, legal Design is an interdisciplinary approach to apply human-centered design to prevent or solve legal problems. The legal design manifesto is available on-line (LeDA 2021). On the other hand, "smart contract" means the specific use of the use of software code to formulate, check and enforce an agreement between contracting agents (Salmerón-Manzano and Manzano-Agugliaro 2019).

The fifth cluster is centered on Law firms as evidenced by the main keywords involved: Law firms, legal technique, legal technology. Law firms are increasingly adopting digital technologies to make their work more efficient as opposed to traditional work methods. The business model of many law firms, like the legal professions as a large whole, will face a significant paradigm shift, as the work provided by law firms in the form of billable hours largely consists of services that do not require higher legal training, but involve mere data processing (Kerikmäe et al. 2018).

The sixth cluster is focused on Legal Education as evidenced by the main keywords involved: Legal education, legal regulation, vocational training. Anticipated changes in the training needs of lawyers and solicitors present a challenge to law schools to revise their curricula (Ryan 2020). Thus, as the legal profession begins to seriously deploy digital technology in the delivery of services and information, more law schools are including technology education in their curricula (Jackson 2016).

The evolution of the concepts related to Legaltech are shown in Figure 4. The colors represent the evolution over time, with blue being the oldest and red the most modern. It can only be seen in the last two years of the study, observing how it evolves from law firms and rule of law at the beginning of 2018, to artificial intelligence or machine learning,

"

i.e., incorporating technologies from computer science. The most occurrence of Legaltech related terms is mostly in 2019. See for example, legal profession, professional regulation, legal services, legal regulation, vocational training, access to justice or law students.

"

**Figure 4.** Evolution of subjects related to Legaltech.

Regarding the concept itself, it can be seen that at the beginning it was legal technology, later legal tech, and finally it has been coined as a term in itself Legaltech. Legaltech appears as an author keyword, according to WoS, in 2017, with no previous records found.


**4. Conclusions**

**Author Contributions:** Investigation, E.V. and O.B.; Methodology, E.V. and O.B.; Supervision, H.C. and N.T.; Visualization, E.V.; Writing–Original Draft Preparation, E.V.; Writing–Review & Editing, E.V., H.C. and N.T.

**Funding:** This research has received funding from Horizon 2020 research and innovation program under MSCA-RISE-2017 (No. 778357) and from Research Lithuanian Council project (No 09.3.3-LMT-K-712-08-0003).

**Conflicts of Interest:** The authors declare no conflict of interest.


**1. Introduction**

With the increasing tension in petroleum resources and environmental issues, several bio-based degradable resources have been drawing wide attention in recent years. Poly(lactic acid) (PLA), which is the most promising bio-based degradable material, can be obtained through the polymerization [1] of lactic acid by fermenting plant starch. PLA has broad application prospects in daily lives, packaging industry, and the biomedical field [2–4] due to its good mechanical properties and biocompatibility. However, the high brittleness, low impact strength [5], and high cost of this material limit its development and applications. An effective way to overcome these drawbacks is to add other biodegradable materials to PLA [6]. Blending PLA with starch [7], which is a widely derived

biodegradable material, yields high-performance and low-cost PLA/starch blends and expands the application range of renewable resources.

Starch contains numerous hydroxyl groups and has high polarity. However, when starch and PLA are blended, using the former as a filler cannot improve and even reduces the toughness of the PLA/starch blends. The layered microstructure along the flow direction is formed in the PLA/starch blends through the pressure-induced flow (PIF) process proposed by Zhang et al. [8]. During the compression process, PLA is oriented, whereas starch is deformed. This process increases the uniformity of the thickness of the PLA crystal layer. The layered structure allows the PLA/starch blend to absorb high impact energy, thereby improving the toughness of the material. However, the use of PIF to improve the toughness of the material is limited, and high pressure causes material damage. Small molecular plasticizers [9,10], such as polyethylene glycol (PEG) and citrate esters, can improve the ductility of PLA/starch blend materials but cannot change the immiscibility of PLA and starch. Plasticizers migrate with time and do not stabilize in the blends. Starch can be chemically modified through esterification, oxidation, crosslinking, and grafting [11] to promote the compatibility of the PLA/starch blend materials. To weaken the hydrogen bonding force among the starch molecular chains, the starch particle size or the polarity of the starch surface can be reduced to enhance the interaction force between PLA and starch. Wang et al. [12] prepared core–shell starch nanoparticles (CSS NPs). By esterifying the starch and subsequently polymerizing the emulsion, the CSS NPs utilized starch as the core to ensure that the material achieves a strong rigidity and polyethyl acrylate (PEA), which has a good a ffinity with PLA, as shell. Given the synergistic e ffect of starch and PEA, the PLA/CSS NP blends exhibited excellent toughness. Thielemans et al. [13] modified nanosized single crystal starch particles by using stearoyl chloride and PEG methyl ether. The crystal structure of the modified starch did not change, but the graft crystallized on the surface of the modified starch did. Appropriate grafts can weaken the hydrogen bonding force among starches and reduce the polarity of the starch surface. These changes help improve the compatibility of PLA/starch blends. In addition to the two-step method to modify PLA/starch composite materials, the chemical modification of starch can also be achieved through one-step reactive blending, and at the same time the compatibility of the PLA/starch composite system can be enhanced. Xiong et al. [14] synthesized a bio-based monofunctional epoxy compound Epicard and used it for the reactive compatibilization of PLA/starch composite materials. The epoxy groups on the Epicard can react with starch and form a hydrophobic layer on the surface of the latter, which will enhance the adhesion of the PLA/starch blends and improve the compatibility between PLA and starch. Jariyasakoolroj et al. [15] used chloropropyl trimethoxysilane (CPMS) modified starch (CP-starch) to melt blend with PLA to prepare PLA/starch composites. During the melt-blending process, CP-starch reacted with PLA to form a block copolymer, which acts as a nucleating agent and can significantly increase the crystallinity of the blend and improve the compatibility between PLA and starch. Modifying starch can e ffectively improve its surface polarity and increase the interaction force between starch and PLA. Reactive blending, which is a simple and practical method, can directly establish the compatibility of PLA/starch composite materials.

In this study, PLA/premixed starch (PSt)/PEG/citric acid (CA) blends were prepared through reactive blending to examine the e ffects of CA and PEG on the toughness of PLA/PSt (wt%/wt%, 70/30) blends. CA with high carboxyl underwent an esterification reaction with starch and consequently enhanced the interface adhesion between starch and PLA. As a plasticizer of PLA and starch, PEG e ffectively dispersed in the matrix and enhanced the mobility of the molecular chains. When the PEG that entered the matrix participated in the esterification reaction between CA and starch, a branched/crosslinked polyester was easily formed at the interface. This polyester further improved the compatibility of the PLA/starch blend materials. This PLA/starch composite material uses biodegradable raw materials, and at the same time, a highly filled PLA/starch composite material with excellent performance is obtained through reactive blending. Therefore, this composite material has broad application prospects in the medical field and packaging field.

## **2. Materials and Methods**


**3. Conclusions**

Following disasters, most people experience unpleasant emotions and experiences. Reactions may be a combination of confusion, fear, despair, helplessness, insomnia, physical pain, anxiety and anger, grief, shock, violence and mistrust, guilt and shame, and loss of trust and self-confidence. Psychological first aid provides a safe, comfortable, connected, self-efficacious, empowered, and hopeful environment.


*Edited by Boris Cogan*

The book "Systems Engineering: Practice and Theory" is a collection of articles written by developers and researches from all around the globe. Mostly they present methodologies for separate Systems Engineering processes; others consider issues of adjacent knowledge areas and sub-areas that significantly contribute to systems development, operation, and maintenance. Case studies include aircraft, spacecrafts, and space systems development, post-analysis of data collected during operation of large systems etc. Important issues related to "bottlenecks" of Systems Engineering, such as complexity, reliability, and safety of different kinds of systems, creation, operation and maintenance of services, system-human communication, and management tasks done during system projects are addressed in the collection. This book is for people who are interested in the modern state of the Systems Engineering knowledge area and for systems engineers involved in different activities of the area. Some articles may be a valuable source for university lecturers and students; most of case studies can be directly used in Systems Engineering courses as illustrative materials.

ISBN 978-953-51-0322-6

Systems Engineering - Practice and Theory

Photo by ubrx / iStock


**Author details**

Marcos Antonio Santana Andrade Junior\*, Hugo Leandro Sousa dos Santos, Mileny dos Santos Araujo, Arthur Corrado Salomão and Lucia Helena Mascaro Department of Chemistry, Federal University of São Carlos, São Carlos, SP, Brazil

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. This chapter is 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.


**Mammalian Heme Peroxidases and**  *Mycobacterium tuberculosis*

Amit K. Singh, Nisha Pandey, Mau Sinha, Sujata Sharma and Tej P. Singh *Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India*

#### **1. Introduction**

276 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

Young D, Lathigra R, Hendrix R, Sweetser D, Young RA. (1988). Stress proteins are immune

Tuberculosis (TB) is a lethal infectious disease which is caused by *Mycobacterium tuberculosis*. The alarming rate at which the incidence of bacterial resistance to known antibiotics has been rising is a serious cause of concern. At present, the two well known anti-tuberculosis drugs, isonicotinic acid hydrazide (INH, isoniazid) and pyrazinamide (PZA, pyrazin-2 carboxamide) which are important components of the current course of the first-line TB chemotherapy suffer from increasing bacterial resistance. The other drugs of the combination therapy include rifampicin and ethambutol. It may be noted that both INH and PZA are prodrugs and require specific enzymes to convert them into drugs. INH is activated by a bacterial heme enzyme catalase peroxidase (*Mt*CP) into a free radical form (Scheme I) (Zhang et al., 1992). The structure of unliganded *Mt*CP is known (Bertrand et al., 2004) and detailed information is available about the substrate-binding site and the residues that might be involved in the binding and conversion of INH into a beneficial product. However, a precise mode of binding and the mechanism of action are not yet clearly understood because the structure of INH bound *Mt*CP is not yet determined. On the other hand, PZA is metabolized into its active form pyrazinoic acid (POA) by amidase activity of the *Mycobacterium tuberculosis* nicotinamidase/pyrazinamidase (PncA) (Scheme II) (Konno et al., 1967). Although the crystal structure of pyrazinamidase in complex with POA is known but the structure of the complex with the original compound PZA is not yet determined. Therefore, the mode of binding of PZA with PncA has not so far been revealed. As shown by the crystal structure of the complex of LPO with INH, the binding of INH to lactoperoxidase (LPO) occurs through the distal heme cavity where INH interacts with a conserved water molecule W1 which is hydrogen bonded to ferric iron (Singh et al., 2010). Similarly, as revealed by the structure determination of the complex formed between LPO and PZA, PZA has been located in the substrate-binding site and interacts with substrate recognition residues of LPO (PDB ID: 3R4X) indicating a possible role of LPO in the conversion of PZA into an active form. Although the crystal structure of the PZA bound PncA is not known but a piece of information is available on the possible mode of ligand binding based on the molecular modeling data (Petrella et al., 2011). Therefore, it is of great interest that both prodrugs, INH and PZA bind to LPO specifically at the substrate-binding site on the distal heme side as the substrates bind to LPO (Singh et al., 2009) so that these

Mammalian Heme Peroxidases and *Mycobacterium tuberculosis* 279

primary function of LPO is to catalyze the bielectronic oxidation of pseudohalide (SCN- ion)

The biological significance of lactoperoxidase is related to its involvement in the natural host defense system against invading micro-organisms (Reiter & Harnulv, 1984; Reiter & Perraudin, 1991; Wolfson & Sumner, 1993). Apart from that, it was also reported to be involved in the antiviral activity (Mikola et al., 1995; Pourtois et al., 1990; Shin et al., 2005), degradation of various carcinogens and protection of animal cells against peroxidative effects (Tenovuo et al, 1985). It may be noted that the reaction products generated by the catalytic

Lactoperoxidase is a heme-containing single chain protein with 595 amino acid residues. Its molecular mass is approximately 68 kDa. LPO is a basic protein with an isoelectric point of 8.2. The carbohydrate content of this protein molecule is about 10% for the four glycosylation sites (Carlstrom, 1969). LPO contains a covalently linked prosthetic group in the catalytic centre which is a derivative of protoporphyrin IX (Thanabal & La Mar, 1989). The iron content of LPO is 0.07% (Paul & Ohlsson, 1985) corresponding to one iron atom per LPO molecule which is a part of the heme prosthetic group. The overall molecular structure of LPO is stabilized by a calcium ion which is strongly bound to LPO molecule through

LPO catalyzes a set of reactions where the resting ferric enzyme (Fe3+) is oxidized rapidly by hydrogen peroxide to form compound I (Kussendrager & van Hooijdonk, 2000), an oxyferryl porphyryl radical species where an oxygen is coupled by a double bond to the iron (Dolphin et

LPO + H2O2 Compound I + H2O (1)

Compound I + S Compound II + S\* (2)

Compound II + S LPO + S\* + H2O (3)

al., 1973) which subsequently oxidizes two aromatic substrate molecules as follows:

Where S is an aromatic substrate and S\* is an 1-electron oxidized form of substrate.

Lactoperoxidase folds into an oval-shaped structure which is largely -helical with 20 helices and two small anti-parallel β-strands (Figure 1) (Singh et al., 2008). The central core of the protein consists of five long -helices, H2, H5, H6, H8 and H12. The N-terminal

action of lactoperoxidase are harmless to mammalian cells (Reiter & Harnulv, 1984).

 ions) **(**Oram & Reiter, 1996; Hoogendoorn et al., 1977) at the expense of hydrogen peroxide (H2O2) in order to generate reactive products with a wide range of antimicrobial activities (Reiter & Harnulv, 1984; Reiter & Perraudin, 1991; Wolfson & Sumner, 1993). LPO also catalyzes the bielectronic oxidation (by two 1-electron steps) of a number of physiologically relevant aromatic organic compounds (Ciaccio et al., 2004; Zhang & Dunford 1993; Monzani et al., 1997; Metodiewa et al., 1989; Metodiewa et al., 1989; Ferrari et al., 1993; Doerge & Decker, 1994; Sipe, 1994; Cavalieri et al., 1997; Ghibaudi et al., 2000;

ion) or hypohalides (OI-

, OBr-

and

) to pseudohypohalide (OSCN-

or halides (I-

OCl-

, Br-

Ramakrishna et al., 1993).

seven-fold coordination.

**2.1 Mechanism of action**

**3. Structure of lactoperoxidase**

and Cl-

compounds are converted into useful antimicrobial products. Since LPO is able to bind and oxidize both of these compounds, the role of LPO in the treatment of TB appears to be quite plausible. It may be mentioned here that the peroxidase activity of *Mt*CP was shown to be associated with the activation of isoniazid (Zhang et al., 1992). It may also be mentioned here that the role of LPO has already been demonstrated in the bacterial clearance of airways by inhaling INH because LPO and H2O2 are present in the mucus of airways (Sawatdee et al., 2006). Thus, understanding the mode of binding of INH and PZA to LPO as well as the mechanisms of action of LPO with respect to these compounds will provide important insights on the possible mode of bindings of INH and PZA to bacterial enzymes *Mt*CP and PncA respectively.

**Isoniazid Isonicotinoyl radical**

Scheme 1.

**Pyrazinamide Pyrazinoic acid**

Scheme 2.

#### **2. Lactoperoxidase**

Lactoperoxidase (EC.1.11. 1.7) (LPO) belongs to the family of mammalian heme peroxidases which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). LPO is present in exocrine secretions such as milk, saliva and tears. Although it is produced at different sites in human body by various glands such as mammary, salivary and lachrymal with varying amino acid sequences but these were found to be chemically and immunologically similar (Kussendrager & van Hooijdonk, 2000). The

compounds are converted into useful antimicrobial products. Since LPO is able to bind and oxidize both of these compounds, the role of LPO in the treatment of TB appears to be quite plausible. It may be mentioned here that the peroxidase activity of *Mt*CP was shown to be associated with the activation of isoniazid (Zhang et al., 1992). It may also be mentioned here that the role of LPO has already been demonstrated in the bacterial clearance of airways by inhaling INH because LPO and H2O2 are present in the mucus of airways (Sawatdee et al., 2006). Thus, understanding the mode of binding of INH and PZA to LPO as well as the mechanisms of action of LPO with respect to these compounds will provide important insights on the possible mode of bindings of INH and PZA to bacterial enzymes

*Mt***CP / LPO**

**Isoniazid Isonicotinoyl radical**

**Pyrazinamide Pyrazinoic acid**

Lactoperoxidase (EC.1.11. 1.7) (LPO) belongs to the family of mammalian heme peroxidases which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). LPO is present in exocrine secretions such as milk, saliva and tears. Although it is produced at different sites in human body by various glands such as mammary, salivary and lachrymal with varying amino acid sequences but these were found to be chemically and immunologically similar (Kussendrager & van Hooijdonk, 2000). The

**PncA**

**N**

**N C**

**O**

**OH**

**C**

**O**

**N**

*Mt*CP and PncA respectively.

Scheme 1.

Scheme 2.

**2. Lactoperoxidase**

**O NH**

**NH2**

**N**

**N C**

**O**

**NH2**

**N**

primary function of LPO is to catalyze the bielectronic oxidation of pseudohalide (SCN- ion) or halides (I- , Br and Cl- ) to pseudohypohalide (OSCN ion) or hypohalides (OI- , OBr and OCl ions) **(**Oram & Reiter, 1996; Hoogendoorn et al., 1977) at the expense of hydrogen peroxide (H2O2) in order to generate reactive products with a wide range of antimicrobial activities (Reiter & Harnulv, 1984; Reiter & Perraudin, 1991; Wolfson & Sumner, 1993). LPO also catalyzes the bielectronic oxidation (by two 1-electron steps) of a number of physiologically relevant aromatic organic compounds (Ciaccio et al., 2004; Zhang & Dunford 1993; Monzani et al., 1997; Metodiewa et al., 1989; Metodiewa et al., 1989; Ferrari et al., 1993; Doerge & Decker, 1994; Sipe, 1994; Cavalieri et al., 1997; Ghibaudi et al., 2000; Ramakrishna et al., 1993).

The biological significance of lactoperoxidase is related to its involvement in the natural host defense system against invading micro-organisms (Reiter & Harnulv, 1984; Reiter & Perraudin, 1991; Wolfson & Sumner, 1993). Apart from that, it was also reported to be involved in the antiviral activity (Mikola et al., 1995; Pourtois et al., 1990; Shin et al., 2005), degradation of various carcinogens and protection of animal cells against peroxidative effects (Tenovuo et al, 1985). It may be noted that the reaction products generated by the catalytic action of lactoperoxidase are harmless to mammalian cells (Reiter & Harnulv, 1984).

Lactoperoxidase is a heme-containing single chain protein with 595 amino acid residues. Its molecular mass is approximately 68 kDa. LPO is a basic protein with an isoelectric point of 8.2. The carbohydrate content of this protein molecule is about 10% for the four glycosylation sites (Carlstrom, 1969). LPO contains a covalently linked prosthetic group in the catalytic centre which is a derivative of protoporphyrin IX (Thanabal & La Mar, 1989). The iron content of LPO is 0.07% (Paul & Ohlsson, 1985) corresponding to one iron atom per LPO molecule which is a part of the heme prosthetic group. The overall molecular structure of LPO is stabilized by a calcium ion which is strongly bound to LPO molecule through seven-fold coordination.

#### **2.1 Mechanism of action**

LPO catalyzes a set of reactions where the resting ferric enzyme (Fe3+) is oxidized rapidly by hydrogen peroxide to form compound I (Kussendrager & van Hooijdonk, 2000), an oxyferryl porphyryl radical species where an oxygen is coupled by a double bond to the iron (Dolphin et al., 1973) which subsequently oxidizes two aromatic substrate molecules as follows:

LPO + H2O2 Compound I + H2O (1)

$$\text{Compound I} + \text{S} \to \text{Compound II} + \text{S}^\* \tag{2}$$

$$\text{Compound II} + \text{S} \rightarrow \text{LPO} + \text{S}^\* + \text{H}\_2\text{O} \tag{3}$$

Where S is an aromatic substrate and S\* is an 1-electron oxidized form of substrate.