*2.6. Multiplex Analysis of Cytokines, Chemokines and Growth Factors in Blood (Serum) and Cerebrospinal Fluid (Liquor) Samples of Experimental and Intact Animals*

Granulocyte colony-stimulating factor (G-CSF), granulocyte and monocyte colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-12 (IL-12), and VEGF are considered cytokines with neuroprotective action, while interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-17 (IL-17), and tumor necrosis factor-α (TNF-α) can cause secondary death of nerve cells. In the present study, preliminary analysis of endogenous cytokines, chemokines and growth factors in cerebrospinal fluid in animals from the control group NaCl showed no significant differences in comparison with intact rats, which probably indicates the cessation of inflammatory response 21 days after modelling a stroke. In addition, intrathecal injection of a combination of adenoviral vectors carrying cDNA encoding VEGF, GDNF, and NCAM, or genetically-modified UCB-MC+Ad5-VEGF-GDNF-NCAM on day 21 of the experiment does not reliably affect the cytokine levels in liquor samples when compared with intact rats.

#### **3. Discussion**

It is known that ischemic stroke leads to massive loss of neurons and glial cells immediately after acute arterial occlusion. Neuron death caused by necrosis and apoptosis is the result of blood flow impairment, oxidative stress, mitochondrial dysfunction, and excitotoxicity. Unfortunately, these irreversible changes rapidly spread beyond the epicenter of the ischemic damage, resulting in progressive neurodegeneration on the following days, and even over the following week [32]. However, during the first hours, an ischemic penumbra with blood supply below a normal level is formed around the ischemic core with irreversible changes. The viability of cells in the penumbra is limited to a 3–6-h period of the therapeutic window, during which it is possible to restrain the entry into apoptosis of neurons and glial cells and thus prevent the increase of infarction volume.

Despite the improved efficiency of modern therapy for ischemic brain stroke, recovery of patients is not satisfactory. Today, specific therapy for acute stroke is aimed at restoring blood circulation in the ischemic area and maintaining the metabolism of brain cells. However, the drugs used in practical medicine are not effective in controlling the death of nerve cells in the ischemic penumbra, which results in an extensive increase in brain infarction. It is known that there are natural limitations neuroregeneration in the CNS; therefore, there is a need to develop new effective approaches to maintain the viability of nerve cells in the ischemic penumbra in the 3–6-h therapeutic window.

It is known that neuroontogenesis (addressed migration of brain cells, directed axon growth and establishment of intercellular contacts) involves an information exchange between neurons, which is realized through neurotrophic factors produced in some neurons or in non-neural cells that affect other neurons. In postnatal ontogenesis, there is no formation of new cortical neurons. Therefore, dying neurons are not restored. This does not mean that there is no regeneration in brain cortex; however, as regeneration is carried out due to intracellular regeneration of damaged neurons, growth of their neuritis and restoration of synaptic connections between regenerating and intact neurons. During neuroregeneration, neurotrophic factors support neuronal survival, stimulate axonal growth, and establish lost synaptic contacts.

At present, the most promising of the actively developed strategies to prevent brain cell death in penumbra is the development of gene and cellular technologies. Gene therapy is mainly aimed at the delivery of therapeutic genes encoding neurotrophic factors. Among them, the most promising are genes encoding neurotrophic factors (BDNF, CNTF, GDNF, VEGF), anti-apoptotic proteins (Bcl-2, Bcl-XL), heat shock proteins (Hsp25, Hsp70), and anti-inflammatory molecules (IL-1RA). The neuroprotective effect of these factors has been experimentally proven, but there is no unequivocal, let alone exhaustive, answer to the question of which of these factors may be recommended as neuroprotective factors in practical medicine. It has been established that combinations of several neurotrophic factors may have a more pronounced effect on nerve cell survival [25,28].

Other equally important issues in the strategy for the gene therapy of stroke include the development of technology to deliver transgenes to the brain. Difficulties in the delivery of therapeutic genes to the cerebral infarction area are one of the main reasons for the unavailability of effective gene therapy in treating post-ischemic negative consequences in the brain. Direct (in vivo) gene therapy provides for the delivery of transgenes into the recipient brain plasmid or viral genetic vectors [33]. Cell-mediated (ex vivo) gene therapy is based on the delivery of therapeutic genes using cells that serve as carriers of transgenes, as well as producers of recombinant protein molecules.

For the treatment of stroke, various methods of gene therapy are known, the effectiveness of which has been proven in numerous experiments on animals [2,4,34]. Injections of viral vectors carrying transgenes into the ventricles or the infarction area are mainly used to deliver the therapeutic genes to the brain. Genetic vectors based on Sendai virus vectors containing *gdnf* or *ngf* genes were injected 30 min after stroke simulation [35]. The adeno-associated viral vector carrying *gdnf* was injected 2 days after the stroke modelling [36]. In other studies, viral vectors carrying therapeutic genes were delivered before modelling the stroke. The positive effect was shown after local delivery to the brain of *gdnf* or *cntf* genes—7 days [37], *bdnf*—2 weeks [38], *gdnf*—4 weeks [39], or *ngf* and *bdnf*—4 to 5 weeks [40] before stroke modelling.

The list of genes employed in gene therapy for stroke is quite long. Of these, we find *vegf*, *gdnf*, and *ncam* to be the most promising.

In addition to angiogenic action, VEGF exhibits the properties of a typical neurotrophic factor. It supports survival of sensitive [41] and motor neurons [42] and stimulates proliferation of astrocytes [43], neural stem cells [44], and Schwann's cells [41]. GDNF has a pronounced neuroprotective effect on dopaminergic brain neurons and cholinergic spinal cord motoneurons [45] and stimulates the growth of nerve processes [46]. NCAM (CD56) is expressed on the surface of neurons and glial cells. Intercellular interactions mediated by NCAM in neuro-ontogenesis and posttraumatic regeneration provide survival and migration of neurons, directed neurite growth and synaptogenesis.

The efficiency of cell therapy for stroke treatment in experiments using neural precursors derived from embryonic stem cells [47], induced pluripotent cells [48], MSCs isolated from red bone marrow [49] or UCB [50] suggests the use of these cells as carriers of therapeutic genes for delivery to the brain. Thus, MSCs were used for delivery of *bdnf* [51], *pigf* [52], and *vegf165* [53] to the brain. Of particular interest is the transduction of cell carriers by two or more expression vectors [28]. This approach allows simultaneous overexpression of several molecules/stimulants of neuroregeneration to be obtained. In our studies, in addition to the gene encoding neurotrophic factors, a gene encoding NCAM was delivered into UCB-MC, which, according to the obtained data, promoted the addressed migration of transplanted cells into the CNS after intravenous injection, increased their survival in the recipient's tissues and supported prolonged production of recombinant therapeutic molecules [28]. In clinical investigations, autologous cells isolated from the red bone marrow (mononuclear and MSCs) or peripheral blood (CD34+) are predominantly used in the cell therapy of patients after stroke [54,55].

The most promising cell carriers of therapeutic genes are UCB-MC [56–58]. The basis for their application is the suitability for both allografting and autotransplantation in humans, availability, and the ease of obtaining and storage. An important factor is the absence of legal, ethical, and religious prohibitions related to blood cell transplantation. In our previous study, we showed that intrathecal injection of adenoviral vectors carrying *vegf*, *gdnf*, and *ncam*, or genetically-modified UCB-MC+Ad5-VEGF-GDNF-NCAM, 4 h after stroke modelling in rats, had a positive effect on the morpho-functional recovery of the post-ischemic brain [25]. Adenoviral vectors and genetically-modified UCB-MC with cerebrospinal fluid reached the ischemic area and delivered the production of recombinant VEGF, GDNF, and NCAM, lasting up to 21 days in the experiment.

Other important issues in the strategy for the treatment of ischemic stroke include the development of approaches to enhance the viability of neurons with the threat of a stroke. Patients with transient ischemic attacks, arterial hypertension, atrial fibrillation, disorders of lipid metabolism with high cholesterol and diabetes are at high risk of ischemic stroke. Preventive therapy aimed at increasing the survivability of neurons in at-risk patients may prevent severe post-ischemic consequences in the brain, or improve the outcome of the disease. Currently, in medical practice, measures to prevent stroke are based on the use of anticoagulants and prosthetics of blood vessels. At the same time, the preventive methods that are able to considerably decrease the death of neurons in the "ischemic penumbra" during the 3–6-h "therapeutic window" are unknown. Enhancement of the viability of nerve cells at risk of stroke is also associated with the delivery of therapeutic genes that encode molecules to the brain, which inhibit neuronal death and stimulate neuroregeneration. In this study, for the first time, we propose the approach of preventive gene therapy to improve the viability of brain neurons under threat of ischemic stroke to contain neuronal death in the first hours of a stroke. The use of leucocytes for delivery of therapeutic genes (*vegf165*, *gdnf*, and *ncam1*) in the brain was based on their biological properties. Leucocytes are cells with high secretory and migration potentials, which suggest their exclusive role as cell carriers for addressed delivery and effective expression of transgenes. The results obtained in the study demonstrate that preventive intrathecal adenoviral- or UCB-MC-mediated delivery of *vegf165*, *gdnf*, and *ncam1* results in a reduction of apoptosis and, consequently, the infraction volume. In addition to the decrease in expression of proteins of cellular stress and restraining neuronal death in the area of ischemic damage, we found evidence of the restoration of functional activity of neurons (increase in expression of synaptic proteins), maintenance of myelinization (increase in the number of oligodendrocytes) and an obstacle to astrogliosis development (decrease in the immunopositive areas for astrocytes and microglial cells markers). Importantly, transplantation of gene-modified UCB-MC is safer and more efficacious compared with direct gene therapy. These data are in line with our results using the same gene and gene-cell constructs for ischemic stroke treatment in rats and allow us to conclude that preventive gene therapy may be effective in overcoming the negative consequences of ischemic stroke in the rehabilitation period.

Recently, for personalized ex vivo gene therapy, we suggested the use of gene-modified leucoconcentrate (GML) prepared from patient's peripheral blood and chimeric adenoviral vectors (Ad5/35F) carrying one or a combination of therapeutic genes [59]. Taken together with the concept of GML-therapy and the data of this study, we propose the use of GML carrying *vegf165*, *gdnf*, and *ncam1* for personalized preventive gene therapy in the threat of stroke.

#### **4. Materials and Methods**

#### *4.1. Preparation and Molecular Analysis of Gene-Modified UCB-MC*

Human (*vegf165*, *gdnf*, *ncam1*) and reporter (*gfp*) genes were inserted into recombinant replication-defective adenovirus serotype 5 (Ad5) in the Gamaleya Research Institute of Epidemiology and Microbiology (Moscow, Russia), as described previously [25]. Viral vectors were grown in HEK-293 cell culture, purified by exclusion chromatography and the titres of Ad5-VEGF (2.6 <sup>×</sup> 109 PFU/mL), Ad5-GDNF (1.7 <sup>×</sup> <sup>10</sup><sup>10</sup> PFU/mL), Ad5-NCAM (2.4 <sup>×</sup> 1010 PFU/mL) and Ad5-GFP (1.2 <sup>×</sup> 1010 PFU/mL) were determined in the HEK-293 cell culture using the plaque formation technique.

The fraction of mononuclear cells from UCB was isolated onto a density barrier by the standard technique of sedimentation, seeded in 10 cm culture dishes and simultaneously transduced with three adenoviral vectors at an equal ratio: Ad5-VEGF (1/3)+Ad5-GDNF (1/3)+Ad5-NCAM (1/3) or with Ad5-GFP with MOI of 10, as described previously [25]. To confirm the efficacy of UCB-MC transduction, the gene-modified cells were cultured for 96 h. Transduction with therapeutic genes (*vegf165*, *gdnf*, *ncam1*) was confirmed by a real-time polymerase chain reaction (RT-PCR) and with reporter green fluorescent protein (*gfp*) using fluorescent microscopy, as described previously [57].

For direct (in vivo) preventive gene therapy, a mixture of 2 <sup>×</sup> 107 virus particles in an equal ratio of Ad5-VEGF (1/3)+Ad5-GDNF (1/3)+Ad5-NCAM (1/3) in 20 μL of saline and Ad5-GFP in 20 <sup>μ</sup>L of saline were prepared. For cell-mediated (ex vivo) preventive gene therapy, 2 <sup>×</sup> 106 UCB-MC+Ad5-VEGF+Ad5-GDNF+Ad5-NCAM in 20 <sup>μ</sup>L of saline and 2 <sup>×</sup> 106 UCB-MC+Ad5-GFP in 20 μL of saline were prepared.

The efficiency of UCB-MC genetic modification by adenoviral vector, bearing green fluorescent protein reporter gene, was analyzed 72 h after cell transduction. The production of green fluorescent protein UCB-MC+Ad5-GFP was examined with the Axio Oberver Z1 inverted fluorescence microscope (Carl Zeiss, Germany). The number of transduced UCBC was estimated using a BD FACSAria III flow cytomography fluorimeter (BD Bioscience, New York, NY, USA) and BD FACS Diva7 software (BD Bioscience, New York, NY, USA).

mRNA levels of *vegf165*, *gdnf*, and *ncam1* transgenes in the UCB-MC, simultaneously transduced by a combination of three adenoviral vectors Ad5-VEGF, Ad5-GDNF and Ad5-NCAM, were analyzed with PCR-RT. Extraction of common RNA from UCB-MC+Ad5-VEGF-GDNF-NCAM and UCB-MC+Ad5-GFP, 72 h after cell incubation, was carried out using a set Yellow Solve (Silex), according to the manufacturer's instructions. Reference samples of common RNA were derived from non-transduced UCBC. The cDNA synthesis was performed using six nucleotide random primers and reverse transcriptase RevertAid Reverse Transcriptase (Thermo Fisher Scientific). Quantitative level analysis mRNA of the target cDNA was performed using a CFX 96 Real-Time PCR System

thermocirculator (BioRad, Hercules, CA, USA), using the TaqMan system. Sequences of primers and probes of the reaction mixture are presented in Table 1. Received data was normalized by the 18S rRNA reference gene. For each target gene, the results were obtained in two independent experiments and presented as a mean value ± SE, *p* < 0.05.


**Table 1.** Experimental groups of animals.

Supernatant obtained 72 h after incubation of gene-modified UCB-MC (UCB-MC+Ad5-VEGF -GDNF-NCAM and UCB-MC+Ad5-GFP) and intact UCB-MC were used for multiplex profiling. In this work, commercially available panels, Human Cytokine/Chemokine Magnetic Bead Panel (HCYTMAG-60K-PX41), were applied, containing the following analytes: EGF, FGF2, Eotoxin, TGF-a, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNa2, IFN-g, GRO, IL10, MCP-3, IL12-p40, MDC, IL12-p70, PDGF-AA, IL-13, PDGF-AB/BB, IL-15, sCD40L, IL-17a, IL-1ra, IL-1a, IL-9, IL-1bIL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP10, MCP-1, MIP-1a, MIP-1b, RANTES, TNFa, TNFb, and VEGF (Millipore). The quantitative analysis of target molecules was performed on a Bio-Plex200 System (BioRad, USA) according to the protocol recommended by the manufacturer and using Bio-Plex Manager 4.1 software (Bio-RadLaboratories). The results were statistically processed using the following methods, using linear models implemented in the limma package. Fold Change was used as an estimate for the effect size, obtained for pairwise comparison.
