Treatment of Dyushen muscular dystrophy with application of fetal progener cell transplantation

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Treatment of Dyushen muscular dystrophy with application of fetal progener cell transplantation

Radchenko V.V., Sirman V.M., Duzhar V.M., Gozhenko A.I.

KRS - Medical Technologies LLC (Kiev, Ukraine)

Ukrainian SRI of Transport Medicine of the Health Ministry of Ukraine (Odessa, Ukraine)

Summary/Резюме

duchenne muscular dystrophy stem cell

The authors are reviewing issues arising in the process of treatment of Duchenne muscular dystrophy (DMD) - a recessive X-linked disorder. This study focused on the effectiveness of the developed DMD treatment method.

Results. The study included 35 patients with DMD aged from 5 to 19 who underwent fetal stem cell transplantation.

Laboratory tests before the treatment were remarkable for significant elevation of CPK in all the patients. Many patients also had high levels of ALT, ACT and LDH.

Administration of stem cells directly into the affected muscles (intramuscularly) results in higher muscle tone, muscle bulk growth stimulation, muscle power and physical capacity increase, immune boosting, improved cognitive and intellectual skills and psycho- emotional state in general.

Key words: Duchenne muscular dystrophy, stem cells, fetal progenitor cells, myoblasts, multi-point intramuscular administration.

Аннотация

duchenne muscular dystrophy stem cell

Авторы рассматривают проблемы, возникающие в процессе лечения мышечной дистрофии Дюшенна (МДД) - рецессивного Х-сцепленного расстройства. Данное исследование было сосредоточено на эффективности разработанного метода лечения МДД.

Полученные результаты. В исследование были включены 35 пациентов с МДД в возрасте от 5 до 19 лет, которым была проведена трансплантация эмбриональных стволовых клеток.

Лабораторные тесты до лечения были отмечены значительным повышением КФК у всех пациентов. Многие пациенты также имели высокий уровень ALT, ACTи LDH.

Введение стволовых клеток непосредственно в пораженные мышцы (внутримышечно) приводит к повышению мышечного тонуса, стимуляции роста мышечной массы, увеличению мышечной силы и физических возможностей, повышению иммунитета, улучшению когнитивных и интеллектуальных навыков и психоэмоционального состояния в целом.

Ключевые слова: мышечная дистрофия Дюшенна, стволовые клетки, клетки- предшественники плода, миобласты, многоточечное внутримышечное введение.

Анотація

Автори розглядають проблеми, що виникають в процесі лікування м'язової дистрофії Дюшенна (МДД) - рецесивного Х-зчепленого розладу. Дане дослідження було зосереджено на ефективності розробленого методу лікування МДД.

Отримані результати. У дослідження були включені 35 пацієнта з МДД у віці від 5 до 19 років, яким була проведена трансплантація ембріональних стовбурових клітин.

Лабораторні тести до лікування були відзначені значним підвищенням КФК у всіх пацієнтів. Багато пацієнтів також мали високий рівень ALT, ACTі LDH.

Введення стовбурових клітин безпосередньо в уражені м'язи (внутрішньом'я- зово) призводить до підвищення м'язового тонусу, стимуляції росту м'язової маси, збільшення м'язової сили і фізичних можливостей, підвищенню імунітету, поліпшенню когнітивних і інтелектуальних навичок і психоемоційного стану в цілому. Ключові слова: м'язова дистрофія Дюшенна, стовбурові клітини, клітини-попе- редники плода, міобласти, багатоточковевнутрішньом'язове введення.

Rationale

Duchenne Muscular Dystrophy (DMD) is a hereditary X-linked recessive disorder caused by abnormal dystrophin synthesis due to genetic defect and resulting in progressive muscular degeneration [1].

According to different sources, DMD affects around 1 in 4, 000 - 6,000 people internationally [2]. On the average, the disease is diagnosed at the age of 3-5 when physical capacity of the affected child is markedly different from that of healthy peers[3]. It is believed that during the first years of DMD patient life his/her muscle fibers regenerate by means of own stem cells of the muscle differon the reserve of which gradually depletes, which leads to abnormal dystrophin production causing muscle degeneration and fibrosis [4].

Rapidly progressive muscle weakness results in gait problems in teenage years. Wheelchair is usually needed by the age of 9-11, but each case is individual. Apart from the progressive muscle weakness, more that 50% of the patients have dystrophin deficiency- induced cardiovascular issues by the age of 15 [5]. In patients aged 20 and older, diaphragm and muscles regulating lung function weaken significantly, therefore they can die from respiratory failure [6]. Gastrointestinal and excretory systems, as well as intellect, are also affected [7].

Over the last years, stem cells are used for DMD treatment [8]. In our opinion, stem cells isolated from fetal muscles can differentiate into myocytes, which suggests that stem cell therapy can be effective in DMD.

The authors are of the opinion that the proposed approaches to treatment will result in higher quality of life and longer life span of DMD patients.

The goal of work is to study clinical effectiveness of the elaborated method of fetal progenitor cell transplantation in DMD.

Materials and Methods

The study included 35 male patients with DMD aged from 5 to 19 examined before and after fetal stem cell (FSCT).

Stem cells were isolated in the Em- ProCell (Mumbai, India) biotechnological laboratory in accordance with international GMP standards.

Stem cells were isolated at the time of organogenesis (beginning stages of muscle system formation) and thoroughly tested for biological safety, aerobic and anaerobic microorganisms, and fungi. Screening also included real time PCR for 12 types of bacteria, genetic testing and karyotyping (XY) [9].

In the course of research, treatment method based on application of fetal progenitor cells and fetal tissue extracts aimed at dystrophin production deficit compensation has been developed. The underlying principle of treatment is transfer of normal (healthy) genetic information of the fetal myoblasts cell nucleus into patient's muscle. Implantation of nuclei of fetal progenitor cells with normal genes encoding synthesis of all 79 exons of dystrophin [10] results in restoration of dystrophin production. Inhibition of muscle tissue degeneration gives time for repeated transplantations of fetal stem cells, which, at the end, results in longer life expectancy and higher life quality in most DMD patients.

Stem cells were administered according to our developed method that included transplantation of two types of allogenic fetal progenitor cells from the same fetus: hematopoietic cells of fetal liver for immune tolerance induction thank to which fetal myoblasts (allogenic muscular cells) administered by multiple intramuscular injections at the next stage are treated by the body like its own [11].

For optimal clinical result, we performed additional subcutaneous administration of fetal myoblasts and fetal placenta extracts containing cytokines stimulating growth and differentiation of both patient's own and transplanted fetal stem cell.

Our method resulted in positive results in Duchenne and myotonic muscular dystrophies as well as in myositis and motor neuron disease.

As a part of the study that lasted for 5 years, many DMD patients repeated stem cells therapy aimed at dystrophin production deficit compensation.

Results and Discussion

For optimal clinical effect in DMD, combination of stem cells and fetal tissues extracts is selected individually for each case of DMD and its complications. This treatment results in the following:

¦ inhibition of the disease progression (longer period of independent ambulation etc.)

¦ preservation of muscle and physical power

¦ gait quality improvement (in walking patients)

¦ improvement or restoration of some skills (climbing stairs, combing, getting up from the floor or sitting position)

¦ reduction of pseudohypertrophy or muscle straining

¦ decreased values of ALT, CPK and LDH signifying subsidence of inflammation in the muscle tissue

¦ prevention or subsidence of the symptoms of myopathy complications

¦ improved function of internal organs and systems

¦ intellect and psycho-emotional state improvement, higher self-esteem

¦ immune boosting

¦ life quality improvement

Table 1 MuscularDystrophyFunctionalRatingScale

The results of treatment with feta myoblasts are different in each patient

Laboratory tests performed before the treatment demonstrated marked CPK elevation in all the patients. Many patients also had elevated ALT and LDH. Functional condition was evaluated on Muscular Dystrophy Functional Rating Scale (MDFRS) (Table 1) [12, 14].

We performed mathematical analysis of DMD treatment effectiveness.

The preliminary analysis demonstrated significant differences in both baseline functional/biochemical parameters and their changes 3 and 6 months after FSCT in the study participants.

First treatment effectiveness evaluation was based on general functional status changes. In 4 patients, MDFRS score increased from 48,7±1,8 to 58,8±1,8 within 3 months and then to 64,0±2,9 within 6 months (Fig. 1). Net differences were +10,0± 1,6 і +15,3± 1,9 accordingly, which gives us reason to regard such pattern as rapidly progressive. Progressive general

Fig.1 Four patterns of individual changes in general functional status by MDFRS: rapidly progressive (rp), slowly progressive (sp), stagnant (s) and reverse (r). In each, triad, the first column is baseline, second - results 3 months after FSCT and third - results 6 months after FSCT. All patients are coded.

MDFRS scoreincreasedfrom 43,6±0,6 to 47,1 ±0,6 andto 51,4±1,0 accordingly, whilenetdifferenceswere +3,5±0,4 and +7,8±0,7 accordingly. Unfortunately, in 10 patients MDFRS scorethatincreasedto4,6±1,6 (from 39,5±0,8 to 44,1±1,8 ) with in first three months remain practically unchanged in the course of the following three months - 43,3±1,8, which was regarded as stagnation. In another 5 children, the first elevation from 39,8±1,3 to 47,4±2,1 was followed by the decreased to 44,6±2,1, and this pattern was regarded as reverse.

Fig. 2. Correlation between CPhK, LDH and ALT changes (X) and MDFRS (Y) 3 months after FSCT.

Fig. 3. CorrelationbetweenCPhK, LDH and ALT changes (X) and MDFRS

(Y) 6 monthsafter FSCT

Correlation analysis demonstrates that general functional status improvement goes parallel with reduced activity of plasma enzymes, or their re(Y^g₆³_m^Cn^rhs atoFSCTCPhK, LDH and ALT changes (X) and MDFRSduced release from myocytesinto the blood. It is interesting functional status improvement, though to note that 3 months after the slow, was also reported in 16 children: treatment, MDFRS score changes are by ALT andCPhKchangeswithoutsignificant LDH change (Fig.2), while 6 monthsafter FSCT CPhKimportancesignificantlyincreases, whiledeterminationimportanceof LDH increasestothatof ALT, whichremainsunchanged (Fig.3).

Table 2 Discriminant Function Analysis Summary

Step 6, N of vars in model: 6; Grouping: 4 grpsWilks' Lambda: 0,066; approx. F(₁₉₎=6,63; p<10^-6

Table 3Summary of Stepwise Analysis

Forvisualization and creation of the coherentenzy mepicture ofeachpattern, for wardstepwisedis criminant function analysis ofequally determined the available data was performed [Klecka WR, 1989] [13]. The program chose 6 parameters (their changes) (D) to be included into the model, while the remaining 2 were not in the model (Tables 2 and 3).

After this, 6-D space of discriminant variables is transformed into 3-D space of canonical roots, which are linear combination of discriminant variables.

Fig. 4. Individual values of roots 1 and 2 with condensed information about MDFRS changes and elevate denzyme sin children with different pattern sofpost-treatment changes.

Discriminative power of the root characterizes canonical correlation coefficient (r*) as a measure of connection and degree of dependency between the groups and discriminant function. For the first root, it equals to 0,919 (Wilks' Л=0,066; X²₍₁₈₎=79; p<10^-6), for the second - 0,667 (Wilks' Л=0,427; x²₍₁₀₎=25; p=0,006), for the third - 0,479 (Wilks' Л= 0,771; x²₍₄₎=8; p=0,110). The other measure of the discriminative power of the root is its percentage amounting to 83,2%, 12,3% and 4,5% accordingly.

Table 4 presents raw (actual) and standardized (scaled) discriminant variables coefficients. Raw coefficient provides information about the absolute contribution of the given variable to the discriminant function value, while standardized coefficients reflect relative contribution of the variable, irrespective of the unit of measure. They allow for detection of variables with max contribution to the value of the discriminant function.

Table 5 presents full structural coefficients - coefficient of correlation between discriminant root and variables. Structural coefficient demonstrates closeness of connection between variables and discriminant functions, or what part of information about discriminant function (root) is stored in the given variable.

Fig. 5. Individualvaluesofroots 1 and 3 withcondensedinformationabout MDFRS changesandelevatedenzymesinchildrenwithdifferentpatternsofpost-treatmentchanges.

The sum of the products of raw coefficients (Table 4) by discriminant variables value (Table 5) with constant (Table 4) dive the value of discriminant function (root) for each child and allow for its visualization in the information field of the roots (Figs.2 and 3).

Table 4

Standardized and Raw Coefficients and Constants for Canonical Variables

As we can see (Fig.4), the extreme left position along the first root axis are children with least favorable functional changes along with minimal CPhK activity changes 6 months after the treatment and minimal LDH changes 3 and 6 months after the treatment. At the same time, delineation of reverse and stagnation pattern representatives is somewhat ill-defined. The extreme right position belongs to the children with progressive improvements, without clear delineation between slow and fast progression. It should be noted that stagnant pattern is delineated along the second root axis by holding the top po- ^Table5sitions, which shows minimal reduction of ALT activity 6 months after the treatment.

Table

Correlations Variables-Canonical Roots, Centroides of Roots and Means of Changes in Variables

At the same time, patients with slow and fast progression are delineated along third root axis demonstrating MD- FRS changes (Fig.5).

Squared Mahalanobis Distances, F-valuesand p-levels

Table Coefficients and Constants for Classification Functions

Generally speaking, delinea- 6tion of all four pat- terns in the information space of the three canonical roots is quite clear, which is documented by calculation of Mahal- anobis distances between cluster centroids (Table 6).

These discriminant parameters can be used for identification (classification) of the patient's belonging to a certain pattern. This goal of the discriminant analysis is fulfilled through the use of classifying (discriminant) functions (Table 7).

Table 8

Classification Matrix Rows: ObservedclassificationsColumns: Predictedclassifications

These functions are special linear combinations maximizing differences between the groups and minimizing dispersion inside them. Classification function coefficients are raw, therefore they are not interpreted.

The object belongs to the group with the maximum value of the function calculated by adding up products of variables value x classification function coefficients + constant.

Table 8 demonstrates practically integral precision of retrospective identification. The data received confirm individual nature of recovery in DMD, but, at the same time, they evidence that they can be divided into four general clusters with two patterns of reaction to SC administration in each - slowly and rapidly progressive.

The parameters obtained also allow for distribution of children into different classes (classification) and prognosis of further course of the disease after stem cell therapy. Thus, the use of the proposed mathematical analysis allows for prognosis and control of individual effectiveness of DMD treatment with stem cells.

Thus, the analysis suggests that stem cell treatment peculiarities and effectiveness depends on individual reaction nature, while MDFRS changes pattern depends on the child's age and other parameters to be studied in the forthcoming research.

The next article will shed light on the possibility of prognosis of the detected patterns of functional and biochemical changes.

Conclusions

1. The proposed method of fetal progenitor cell administration is an effective and promising therapeutic approach in DMD.

2. Administration of stem cells directly into the affected muscles is clinically effective and results in positive biochemical changes.

3. Stem cell therapy effectiveness is, to a great extent, determined by the individual nature of reaction.

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