Optical and active sensor derived variables used in forest AGB estimation.
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More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\\n\\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\\n\\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\\n\\nAdditionally, each book published by IntechOpen contains original content and research findings.
\\n\\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\\n\\n\\n\\n
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'
Simba Information has released its Open Access Book Publishing 2020 - 2024 report and has again identified IntechOpen as the world’s largest Open Access book publisher by title count.
\n\nSimba Information is a leading provider for market intelligence and forecasts in the media and publishing industry. The report, published every year, provides an overview and financial outlook for the global professional e-book publishing market.
\n\nIntechOpen, De Gruyter, and Frontiers are the largest OA book publishers by title count, with IntechOpen coming in at first place with 5,101 OA books published, a good 1,782 titles ahead of the nearest competitor.
\n\nSince the first Open Access Book Publishing report published in 2016, IntechOpen has held the top stop each year.
\n\n\n\nMore than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\n\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\n\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\n\nAdditionally, each book published by IntechOpen contains original content and research findings.
\n\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\n\n\n\n
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Enhancing public health is of significant importance to the development of a nation, particularly for developing countries where the health care system is underdeveloped, fragile or vulnerable.This book examines progress and challenges with regards to public health in developing countries in two parts: Part 1 “General and Crosscutting Issues in Public Health and Case Studies” and Part 2 “Country-Specific Issues in Public Health.” For example, assuring equity for marginalized indigenous groups and other key populations entails the application of transdisciplinary interventions including legislation, advocacy, financing, empowerment and de-stigmatization. The diverse structural, political, economic, technological, geographical and social landscape of developing countries translates to unique public health challenges, infrastructure and implementation trajectories in addressing issues such as vector-borne diseases and intimate partner violence.This volume will be of interest to researchers, health ministry policy makers, public health professionals and non-governmental organizations whose work entails collaborations with public health systems of developing nations and regions.",isbn:"978-1-78985-874-7",printIsbn:"978-1-78985-873-0",pdfIsbn:"978-1-83962-380-6",doi:"10.5772/intechopen.83134",price:119,priceEur:129,priceUsd:155,slug:"public-health-in-developing-countries-challenges-and-opportunities",numberOfPages:274,isOpenForSubmission:!1,isInWos:null,hash:"28c7e86f71905feb65668941c4f259f4",bookSignature:"Edlyne Eze Anugwom and Niyi Awofeso",publishedDate:"September 9th 2020",coverURL:"https://cdn.intechopen.com/books/images_new/9138.jpg",numberOfDownloads:6107,numberOfWosCitations:0,numberOfCrossrefCitations:1,numberOfDimensionsCitations:8,hasAltmetrics:1,numberOfTotalCitations:9,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"March 14th 2019",dateEndSecondStepPublish:"May 2nd 2019",dateEndThirdStepPublish:"July 1st 2019",dateEndFourthStepPublish:"September 19th 2019",dateEndFifthStepPublish:"November 18th 2019",currentStepOfPublishingProcess:5,indexedIn:"1,2,3,4,5,6",editedByType:"Edited by",kuFlag:!1,editors:[{id:"293469",title:null,name:"Edlyne Eze",middleName:null,surname:"Anugwom",slug:"edlyne-eze-anugwom",fullName:"Edlyne Eze Anugwom",profilePictureURL:"https://mts.intechopen.com/storage/users/293469/images/9729_n.jpg",biography:"Edlyne Anugwom is a Professor of Sociology and Development\nat the University of Nigeria and until recently Georg Forster\nSenior Research Fellow of the Humboldt Foundation at the\nInstitute of African Studies, Leipzig University. He has held\nteaching positions/fellowships in such diverse places as Leiden,\nWassenaar, Birmingham, Bridgewater, Edinburgh, Mainz, Cape\nTown, and others. His research interests are in the areas of social\nresearch, public health, political sociology of development, social conflict and terrorism in Africa. Significant publications include: Research Methods in Social Sciences\n(2010, Fourth Dimension Pub); The Boko Haram Insurgence in Nigeria: Perspectives\nfrom Within (2019, Palgrave Macmillan); From Biafra to the Niger Delta Conflict:\nMemory, Ethnicity and the State in Nigeria (2019, Lexington Books); and Development in Nigeria – Promise on Hold? (2020, Routledge). 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\r\n\tFor more than a century, the worldwide control of Tuberculosis remains elusive. Until recently, most approaches to controlling Tuberculosis have centered on biomedical approaches such as drugs and vaccines. Tuberculosis risk and susceptibility is linked to upstream health determinants. It is well established that Tuberculosis is a socio-economic disease whose gradient closely follows the social/economic gradient both within countries and within communities.
\r\n\tIn recent times, many high Tuberculosis burden countries are undergoing health transition with changes in the physical, demographic, political, and socio-economic environments, all of which are influenced by the Tuberculosis disease burden. This book aims to explore the link between Tuberculosis, the environment, host and social related factors and the importance of these factors in crafting a multi-faceted approach required to prevent and manage this complex disease.
The aboveground biomass (AGB) of forests directly provides the amount of organic matter in living and dead plant materials, for example, of leaves, branches, and stem, and it is expressed in dry weight per unit area [1]. AGB is also one of the major reservoirs of carbon in forest ecosystems and a key indicator of forest health [2]. Thus, measuring and monitoring AGB changes is crucial to understanding AGB role in the global carbon cycle to reduce carbon dioxide concentrations and to mitigate climate change [3]. AGB was recognized as an Essential Climate Variable by the United Nations Framework Convention on Climate Change (UNFCCC) [4] and an essential biophysical parameter of forest ecosystems [5] for estimating carbon and water cycling, and energy fluxes between land surface and atmosphere. These are processes relevant on the background of climate change [6]. In addition, AGB resources underlie the development of a bio-based economy as part of the European Union Forest-Based Vision targets sector 2030 [7]. The key role of AGB forests leads to the need for accurate and precise estimates of AGB to assess changes in forest structure, including understanding the roles of forests in the terrestrial carbon flux and global climate change [8].
Spatial and temporal quantification and monitoring of AGB are important to assess the impacts of climate and land use changes on the global carbon cycle and understand the causing effects on ecosystem resilience [5]. Fast, accurate and timely estimation and monitoring of AGB, at local, regional, and global scales, will significantly reduce the uncertainty in carbon stock assessments and provide valuable information to better support sustainable forest management strategies [9, 10]. The frequently used methods to estimate AGB are the indirect, which are based on mathematical relations between biomass, dependent variable and one or a few easy-to-measure tree variables, independent variables [11]. Traditionally, indirect methods use forest inventories and allometric functions at tree level to evaluate biomass at plot level and an extrapolation method to assess all area [12].
In last decade, there has been an increase of biomass mapping and estimations for global terrestrial ecosystems using remote sensing (RS) [6]. Satellite-derived predictor variables have been used to estimate AGB and assess its spatio-temporal variability through valuable RS approaches [13]. The development and implementation of RS-based AGB estimation’ monitoring frameworks may provide low-cost and accurate operational geospatial tools for rapidly and effectively detecting, mapping and assessing relevant changes in AGB in any study area. On the other hand, this same type of geospatial tool might be able to support the execution, monitoring, and impact evaluation of nature conservation policies and strategies, by providing a systematic and accurate forest AGB estimation monitoring system [14]. RS provides variables indirectly related to AGB, and the spectral response (original or transformed sensor bands) of the vegetation cover, density, shade, and texture is correlated with AGB [15].
In response to the urgent need for much improved mapping of global forest biomass and the lack of current space systems capable of meeting this need, the BIOMASS mission arose from European Space Agency (ESA) as an Earth observing satellite planned to launch in October 2022 [16]. BIOMASS mission aims to provide the scientific community with accurate maps of world’s forest biomass, including height, state, disturbance patterns, and how they are changing [17, 18]. Gathering BIOMASS mission information with the economic and environmental knowledge will enable to reach a better spatial planning of the forest AGB [19].
The objective of this chapter is to present the satellite images, satellite-derived predictor variables and algorithms used to estimate forest AGB with RS data. To accomplish this objective, the Section 2 presents the types of satellite sensors used in RS. The Section 3 describes the satellite-derived predictor variables. In Section 4, there is a description of the most used algorithms to predict forest AGB. Section 5 presents a discussion of the more frequently used satellite images, algorithms, and variable importance for AGB estimation with RS. Finally, in Section 6, the main conclusions are presented.
Currently, there is a wide variety of RS data available for forest AGB estimation, such as optical (passive) and radar (active) sensors data, at different spatial, spectral, and temporal scales resolution, from local to global scale, with or without costs [20]. The selection of the proper satellite data it will depend on the scope of the research and on the study area, considering the area size, type of forest, and available budget to obtain accurate forest AGB estimations. Optical and radar RS follow similar approaches for forest AGB analysis, modeling, mapping, and monitoring [21]. Optical RS imagery gives spectral information of the horizontal forest structure [22], while radar RS imagery gives information of the vertical forest structure due to the ability of penetrating the forest canopy to a certain depth [23].
The analyze of optical data is easier to use than radar RS data because, after calibrating and correcting the images, the data can be directly processed and extracted and similarly interpreted as a photograph. As passive sensor, optical RS sensors needs a light source (e.g., sun light), and the quality of the images is dependent of the weather and day light. These sensors record a variety of electromagnetic spectrum radiation frequency, especially at wavelengths of visible light and infra-red. Optical RS sensors record the reflectance of the electromagnetic spectrum of earth objects in the visible (Blue, Green, and Red) and infrared regions (near infrared – NIR and short wave infrared – SWIR). Visible and NIR (VNIR), and SWIR are the wavelengths more sensitive to vegetation characteristics.
Optical RS data are widely used to estimate AGB of different types of forest, such as in temperate forests [24], Mediterranean forests [25], sub-tropical forests [26], and boreal forests [27]. Different types of spatial resolution sensors (low: 100–1000 m, and medium: 10–100 m) have been used in studies of AGB estimation at global, regional, and local scale, such as MODIS [15], Landsat ETM+ [28], SPOT [29], and more recently Sentinel-2 [30]. Very high spatial resolution sensors (<1 m), such as IKONOS, Quickbird II, WorldView-2, have advantage over low and medium spatial resolution sensors, despite their cost. These sensors provide finer spatial scale with a greater thematic resolution and accuracy which can be used to complement AGB forestry measurements from the forest inventory [31, 32]. Monitoring and evaluation of AGB estimation over time can be performed by using optical RS due to existing continuous data and wide spatial coverage. However, despite the widespread usage of optical RS data in the estimation of forest AGB, these are limited to their poor penetration capacity in the forest canopies and clouds, in addition to presenting problems of data saturation in high AGB density [33].
On the contrary, interpretation of radar RS sensor data, that is, Synthetic Aperture Radar (SAR) and Light Detection and Ranging (LiDAR) sensors imagery, is not always straightforward because of the signal being responsive to surface characteristics, like structure and moisture, and consequently to have the non-intuitive and side-looking geometry. Despite that, radar provides much more information than just an image to be visually analyzed because of being characterized by two data values for each pixel, a Magnitude value (image analogous to one collected by an optical sensor) and a Phase value (it cannot be visually interpreted). As an active sensor, radar has the advantage of providing its light source and enabling it to operate 24 hours a day.
SAR sensors have been used to estimate forest AGB to complement the spectral reflectance characteristics of vegetation in the optical regions and are very useful in regions often covered by clouds [34]. These sensors are sensitive to water content in vegetation and register information independently of the weather conditions [35] through the interaction of the radar waves with tree scattering elements [17]. The techniques most used in biomass studies using SAR data are regression based on backscattering amplitudes [36] and interferometry based on backscattering amplitudes and phases [37]. The important factors in the backscattering coefficient are wavelength (e.g. ,K, X, C, L, P), polarization (e.g., HH, VV, HV, VH), incidence angle, land cover, and terrain properties (e.g., roughness and dielectric constant).
The short wavelength X and C bands, which interact only with tree canopy surface layer information, are more suitable for AGB estimation in areas with low biomass [38]. The long wavelength L and P bands are more suitable for AGB estimation of dense forest with relatively high biomass density for presenting greater interaction with the forest elements, such as branch, stem, and soil under the canopy [39] and by allowing canopy height to be retrieved by polarimetric interferometry [17].
The polarization information can be linear and crossed. The linear polarization is obtained through linear transmission and reception signal, horizontal (H) and vertical (V), HH and VV, respectively [40]. In the cross-polarization, the transmission and reception signal are different, i.e., HV and VH. Cross-polarized VH backscatter has been considered the largest dynamic range and the highest correlation with biomass [17]. In the case of linear polarization, ground conditions can affect the biomass-backscatter relationship. HH backscatter comes mainly from stem-ground scattering, while VV backscatter results from both volume and ground scattering.
SAR sensors has been used in several forest AGB estimation studies, such as in Miombo Savanna Woodlands [41], deciduous broadleaved and mixed broadleaf-conifer forests [30, 42], tropical peat swamp forests [43], tropical savannas and woodlands [44], temperate deciduous forest [45], rainforest [46], coniferous temperate forest [47], and mixed and deciduous boreal forest [48]. Currently, there is a considerable number of ongoing SAR missions from various agencies, namely Sentinel-1A,B, ALOS-2, SOACOM-1a,b, Cosmo-SkyMed SG, and PAZ. For historical analysis, it can use data from sensors, such as, ERS-1,2, JERS, Envisat, Radarsat-1,2, ALOS, TerraSAR-X, and TanDEM-X. However, despite the positive correlation with the forest structure parameters, SAR data can present saturation problems. The saturation problems can be over different types of temperate, boreal, and tropical forests [49] and depend on the wavelengths, L band at around 100–150 t/ha [50] and 250 t/ha [51], polarization, characteristics of the vegetation stand structure and ground conditions [52]. Furthermore, estimating AGB with only SAR data is difficult, since these data provide information on the roughness of the land cover surfaces and do not distinguish the types of vegetation [53].
LiDAR is an active RS method sensor composed by a laser, a scanner and a specialized GPS receiver used from spacecraft satellite for Space-borne Laser Scanning (SLS), aircraft for Airborn Laser Scanning (ALS) – the most used in forestry approaches – and on the ground level, Terrestrial Laser Scanning (TLS). LiDAR uses pulsed laser light to measure ranges (variable distances) to the object Earth. Differences in return times and laser wavelengths serve to calculate distance traveled which is then converted to elevation. These measurements generate a cloud of points that allow the 3D representation of the vegetation, based on the identification of the X, Y, and Z location, and can penetrate within forest canopy [54]. The airborne LiDAR has the advantage of covering a large area, allowing its use in large areas with minimum occlusion of the terrain by vegetation. Also, it does not saturate even at very high biomass levels (>1000 Mg/ha) [55].
LiDAR provides accurate measurements of vegetation structures, such as height, crown size, basal area, stem volume, and vertical profile. These measurements to characterize the canopy or crown cover in three dimensions and consequently to estimate forest AGB in any study area [56]. The LiDAR metrics can be extracted on the basis of either individual trees [57] or areas [49]. The extraction of these metrics depends on the laser return signal (discrete-return vs. waveform), scanning pattern (scanning or profiling), and footprint size (small vs. large). LiDAR data have been used to estimate forest AGB in combination with other sensors data (optical and/or radar) [15, 58]. Moreover, LiDAR data are a good complement to forest inventory data because they capture spatial variability and can be acquired quickly and in large or difficult to access areas [58]. However, the limited availability and the cost of these data prevent its extensive application [10].
Integration of multi-source RS data, that is, optical, SAR or/and LiDAR data, is important to improve AGB estimation because more information about forest structure features is integrated than just by a sensor. The integration of multiple data sources for more accurate forest AGB estimations has been explored by several authors [44, 59, 60]. In this way, the advantages of each sensor are highlighted to the detriment of negative characteristics of the sensors [61]. Nevertheless, RS data should be complemented with AGB field data measurements, as training and validation data, in order to improve the accuracy of the AGB estimation model [6].
In studies of forest AGB estimation, it is important to integrate different types of independent variables derived from passive and active sensors, such as original spectral bands, transformed images, vegetation indices, and textural variables (Table 1), to achieve accurate predictive models [62].
Sensor | Variable | Description | References |
---|---|---|---|
Optical | Spectral features | Spectral bands, vegetation indices, and transformed images | (e.g. [62]) |
Spatial features | Textural images | (e.g. [63]) | |
Active | Radar | Backscattering coefficients, textural images, interferometry SAR, and Polarimetric SAR interferometry | (e.g. [38, 64, 65, 66]) |
LiDAR | Lidar metrics based on statistical measures of point clouds or estimated products (e.g. canopy height or individual trees) | (e.g. [67, 68]) |
Optical and active sensor derived variables used in forest AGB estimation.
Spectral bands (e.g., VNIR and SWIR) reflect the vegetation structure, texture, and shadow, related with leaf cellular structure and plant pigments, which are correlated with AGB [15]. VNIR wavelengths are more sensitive to pigments and overall canopy health, while SWIR capture many biochemical, leaf mass per area, and discriminates moisture content of soil and vegetation [15]. In Red region occurs absorption by chlorophyll, while in NIR region, there is a pronounced reflection by mesophyll cells [69]. Green band is related with the greenness of vegetation representing the absorption intensity of Blue and Red energy by plant leaves and the reflection intensity of green energy [70]. On the other hand, hyperspectral sensors allow to have many narrow contiguous spectral bands and can accurately discriminate absorption features’ wavelength position and shape.
Transformed images have been used to reduce the dimension of the data, required for optimal performance, by producing new variables from optical multispectral data [21]. There are some image transformation techniques, such as Principal Component Analysis (PCA), Minimum Noise Fraction transform (MNF), and Tasseled Cap Transform (TCT). PCA is the most used transformed image to enhance the change information from stacked multisensor data and captures maximum variance generating a new reduced set of bands in which the information is concentrated and that have little correlation [71]. For n bands of multispectral data, the first PC (PC1) includes the largest percentage of the total image variance and the succeeding components (PC2, PC3, …, PCn) each containing a decreasing percentage of the scene variance [70].
MNF transform is a linear transformation of the reflectance data of hyperspectral and high spectral resolution images to determine the inherent dimensionality of image data, to segregate noise in the data, and to reduce the computational requirements for subsequent processing [72, 73]. MNF is composed of two PCA rotations that separate the noise from the data. The first rotation consists in the call noise whitening process in which the principal components of the noise covariance matrix are used to decorrelate and rescale the noise in the data and obtain transformed data. The transformed noise data have unit variance and no band-to-band correlations. The second rotation uses the principal components derived from the noise whitening process and rescaled by the noise standard deviation. The inherent dimensionality of the second transformation data is determined by examining the final eigenvalues and the associated images. In this way, MNF transformation may reduce the dimensionality of hyperspectral image data, eliminate correlations band-to-band, and order components in terms of image quality [74].
TCT is a conversion of the original bands of an image into a new set of bands through an orthogonal transformation with defined interpretations, useful for vegetation mapping, and directly associated with important physical parameters of the vegetation [75]. TCT uses a similar concept to the PCA in which linear combinations of the original image bands are performed. The tasseled-cap band are produced as the sum of image band 1 times a constant plus image band 2 times a constant, etc. The coefficients used to create the tasseled-cap bands are derived statistically from images and empirical observations and are specific to each imaging sensor. TCT aims to compress the spectral data in few bands associated with physical scene characteristics with minimal information loss [76]. These bands are then correlated, transforming them orthogonally into a new set of axes associated with physical features. The resulting spectral features consists in three axes defined as brightness, greenness, and wetness that are directly associated with important physical parameters [77]. Brightness – the first feature of TCT – is a weighted sum of all the bands and is related with bare or partially covered soil, natural and man-made features, and variations in topography [70]. Greeneess – the second feature of TCT – is a measure of the contrast between the NIR band and the visible bands and is related with vegetation amount in the image. Wetness – the third feature of TCT – is orthogonal to the first two components and is related to canopy and soil moisture [78]. TCT was developed in 1972 for the Landsat multi-spectral scanner (MSS) to understand the growth patterns of plants, soil moisture, and other hydrological features in the spectral space formed by combinations of different bands but is adapted to current sensors.
Vegetation index is a spectral transformation of at least two bands to improve the contribution of the vegetation properties of an image. The wide variety of vegetation indices are calculated based on the ratio between two or more bands to contrast the high absorption by leaf pigments (chlorophylls, carotenoids, and xanthophylls) in the visible spectral region (400–700 nm), high reflectance by leaves in the NIR region (700–1300 nm), and moderate water absorption in the SWIR (1300–2100 nm) [79]. There is a wide range of vegetation indices that are used in the estimation of AGB (Table 2).
Vegetation indices | Formulation | Reference | |
---|---|---|---|
Normalized difference vegetation index | NDVI | [84] | |
Enhanced vegetation index | EVI | Note: C1 = 6; C2 = 7 5; L = 1; G = 2 5 | [85] |
Modified simple ratio | MSR | [86] | |
Specific leaf area vegetation index | SLAVI | [87] | |
Soil-adjusted vegetation index | SAVI | [88] | |
Triangular vegetation index | TVI | [89] | |
Corrected transformed vegetation index | CTVI | [90] | |
Transformed triangular vegetation index | TTVI | [91] | |
Ratio vegetation index | RVI | [92] | |
Normalized ratio vegetation indexes | NRVI | [93] | |
Infrared percentage vegetation index | IPVI | [94] | |
Optimized soil-adjusted vegetation index | OSAVI | Y = 0.16 | [95] |
Normalized difference index using bands 4 & 5 of Sentinel-2 | NDI45 | [96] | |
Inverted red-edge chlorophyll index (Sentinel-2) | IRECI | [97] | |
Transformed normalized difference vegetation index | TNDVI | [98] | |
Sentinel-2 red-edge position | S2REP | [97] | |
Green normalized difference vegetation index | GNDVI | [99] | |
Simple ratio | SR | [100] | |
Green ratio vegetation index | GRVI | [101, 102] | |
Normalized difference water index | NDWI | [83, 103] | |
Moisture stress index | MSI | [104] |
Vegetation indices used to establish the AGB model.
Two most common vegetation indices used in forest AGB estimation are NDVI and SR [63]. NDVI is the fraction of the difference and the sum of the NIR and Red bands where chlorophyll absorbs Red whereas the mesophyll leaf structure scatters NIR [80]. SR is the ratio between NIR and RED [81] and intends to capture the contrast between the RED and NIR bands for vegetated pixels. Both vegetation indices prove to have a good relation with the AGB estimation derived from satellite images data in several types of forests [63, 82]. Further, canopy moisture content can be quantified through vegetation water indices, namely NDWI which is related with NIR and SWIR bands [83].
Texture is a feature used to identify objects or regions of interest in an image [105], based on mathematical pattern (spatial) analysis. Texture is characterized by defining local spatial organization of spatially varying spectral values that is repeated in a region of larger spatial scale. The variations in these scales in the image values that constitute texture are generally due to an underlying physical variation in the landscape that changes reflectivity or emissivity [106]. Textural analysis techniques can be used to provide quantitative metrics that are highly sensitive to the underlying processes of change [107]. However, as the texture has many different dimensions, there is no single texture representation method that is suitable for a variety of textures. There are several methods of extracting textures from RS images; however, texture measurements based on the gray level co-occurrence matrix (GLCM) [105] is one of the most used in forest AGB estimation [64, 108]. The extraction of appropriate descriptions of texture involves selecting moving window sizes which in GLCM is a key parameter in texture analysis [106, 109]. Theoretically, variation and contrast should increase with increasing size and displacement of the window until the size of the textured objects is reached [108]. Overall, small windows produce noisier estimates of the texture descriptor and maintain a high spatial resolution, while larger windows amplify the estimation errors near spatial instances. Due to the variety of objects in an image, when estimating texture parameters should not be used a fixed window. The estimation of texture parameters should be done based on the directional invariant measures, which are the averages between the texture measures of four directions (0, 45, 90,° and 135°) [110]. There are also 8 statistical texture measures, from 14 suggested by Haralick [105], considered the most relevant for the analysis of RS images: angular second moment, contrast, variance, homogeneity, correlation, entropy, mean, and dissimilarity [64]. The information of each of these texture measures depends on the type of image to be analyzed in relation to the spectral domain, the spatial resolution and characteristics of detected objects (size, shape, and spatial distribution). In addition, when faced with a complex forest structure textural images have stronger relationships with biomass than the original spectral bands [64].
Forest AGB estimated from RS data is usually via an indirect relationship between the spectral response (original or transformed sensor bands) and AGB calculations based on field measurements, allowing an extrapolation of field data collected for larger scales [111, 112]. Different prediction methods can be applied to estimate forest AGB [52, 113]. The most used methods for forest AGB estimation are the linear and multiple regression models [114]. However, in recent years’ machine learning methods, such as, random forest (RF), support vector machines (SVM) and artificial neural network (ANN) have become more prevalent [113].
Linear and multiple regression models are parametric algorithms which assumes that there is a linear relationship between both the dependent (i.e., AGB) and independent (derived from DR) variables [115]. Simple linear regression establishes a relationship between a dependent and one independent variable. If there is a relationship between two or more independent variables, the regression is called multiple linear regressions. Multiple regressions can be linear and nonlinear. This type of regression also allows to determine the relative contribution of each of the independent variables to the total explained variance and the explained variation of the model. Despite being an easy method to calculate the relationship between RS-derived variables and forest AGB, parametric algorithm is not simple global linear because it is affected by many factors (e.g., forest age, tree species, and tree height). Thus, a stepwise regression model might be applied to identify the appropriate RS-derived variables that present strong relations with forest AGB [114].
Among the existing non-parametric algorithms, only the most commonly applied to predict forest AGB estimation using RS data will be described, that is, RF, SVM and ANN [10, 52]. Non-parametric algorithms are more flexible than the parametric algorithms and create more complex models of non-linear AGB. These machine learning methods are a more reliable technique to estimate AGB [8] because do not have predefined model structures and the data determined the structure of the model.
RF is a machine learning classification and regression technique that creates a vast number of uncorrelated decision trees at training time, where the most accurate decision tree can be voted [116]. In addition, regression tree-based methods have a higher potential to identify non-linear relationships between dependent and independent variables [117]. This advantage is significant for RS-based studies, where data have shown low linear relation with AGB and the variables might be collinear [113]. RF has also the advantage of using multisource data in large study areas [10].
SVM is a machine learning algorithm that analyzes data used for classification and regression analysis [118]. This algorithm, from a set of category-identified training examples, build a model in which the new examples are attribute to one category or another. SVM constructs a linear separation rule between examples in a higher-dimensional space induced by a mapping function in training samples. This algorithm has the ability to use small data training samples to produce relatively high estimates of forest biophysical parameters using remote sensing data [10].
ANN is an important non-parametric model for forest parameter estimation [119] that simulates the associative memory as animal brain [120]. This algorithm learns by processing examples that have one or more inputs (independent variables from different data sources, such as RS and ancillary data) and known results, establishing associations by probability that will contribute to the “learning.” These associations are stored in their net data structure. After receiving several examples, the net is able to predict the results from inputs using the previously established associations. Thus, the greater the number of examples, the greater the accuracy of ANN’s predictions will be. However, the relationship between the dependent and the independent variable is not easily interpreted [10].
Accurate predictive models of forest AGB are of great importance for forest management and climate mitigation [121]. In general, there are three widely used methods of validation of forest AGB estimation. According to Lu et al. [10], the first method consists in selecting a set of sample plots through random, systematic, and stratified random sampling. The sample plots will be randomly divided into two subsets. One of the subsets will be used to train the model (e.g., 75% of subset data), while the other will be used to calibrate the model (e.g., 25% of subset data). In this case, both subsets are produced from the same sample plot which may lead to an overestimation of accuracy. The second method is cross-validation where a set of sample plots is selected using one of the first sampling previous methods. Here, a plot sample is removed while the remaining plots are used for the development of forest AGB estimation model. This method has a similar advantage to the first; however, it presents a more reliable precision assessment. The third method involves the use of an independent set of sample plots collected through a sampling design. However, despite being theoretically reliable, this method is more expensive.
These accuracy statistics are often expressed as the coefficient of determination (R2), a measure of how well model predictions explain the target variance of the validation set, and the root mean square error (RMSE), a frequently used measure that indicates the absolute fit of the model to the data (i.e., how close the observed data points are to the model’s predicted values). RMSE is a good measure of how accurately the model predicts the response, and it is the most important criterion for fit if the main purpose of the model is a prediction [122]. In general, a high R2 and a low RMSE value shows a good adjustment between the model developed and the sample plot data. Thus, obtaining an accurate predictive model of forest AGB estimation is important to provide valuable information to better support sustainable forest management strategies to mitigate climate change and preserve the multiple ecosystem services provided by forests.
Over the past decades, there has been an improvement in satellite data from sparse coarse to medium and fine spatial resolutions, allowing better accuracy in estimating forest AGB at local, national, and global scale [69]. Recently with the launch of the Sentinel satellite family, more accurate predictive models of forest AGB estimation may be produced due to the existence of better spatial (bands with 10, 20, and 60 m) and spectral resolutions data, with a 5 days’ revisit time of these satellite family in comparison with other free commercial satellite data, as Landsat or MODIS [59]. For instance, Landsat images with spatial resolution of 30 m contain many mixed pixels, and a pixel can contain different trees species and vegetation ages. In addition, large amount and good quality of field measurements, obtained from forest inventory plots data [123, 124] and/or from LiDAR data [125] should be used, as training and calibration data, to obtain accurate model of forest AGB estimation.
More recently, the studies of forest AGB estimation have been using the combination of optical and radar data. The integration of different remotely detected data sources showed to increase the accuracy of the predictive models of forest AGB estimation. In this way, the incorporation of forest structural parameters of SAR data overcome the problems of mixed pixels and data saturation caused by optical data [30, 126]. For instance, Townsend [127] observed that the model’s performance for estimating biophysical properties of forests has improved due to the capabilities of the Landsat TM and SAR data. On the other hand, Forkuor et al. [44] when mapping forest AGB found better predictive accuracy of AGB when combining optical and SAR sensors (Sentinel-1 and 2) than individually. However, several authors corroborate that optical sensors produce better forest AGB estimation results than SAR when used individually [8, 44, 128] despite the lack of sensitivity of the optical data to AGB beyond the canopy closure and grass interference in savannas and forests [129, 130].
In the last years, predictive models to estimate forest AGB have been applying machine learning algorithms based on decision trees instead of the traditional parametric regression models [59]. Machine learning algorithm (e.g., ANN) showed advantage over regression algorithms for being versatile and flexible [131]. This advantage was observed by Ou et al. [132] when comparing with two parametric models (linear regression model and linear regression with combined variables), the two non-parametric models (RF and ANN) resulted in significantly greater estimation accuracies of forest AGB, that is, higher coefficient of determination (R2) and lower root mean square error (RMSE). Other authors corroborate with this statement by showing that non-parametric models have greater capacity to better capture the heterogeneity of forest AGB compared to parametric models [47, 64, 128, 133].
Among the variety of machine learning techniques, RF algorithm revealed to be one of the best methods for classification and regression by providing high accuracy in estimating forest AGB, high speed of computation, robustness and capacity to predict the important variables either using optical or SAR data [30, 59, 128, 134, 135, 136, 137]. Also, RF showed to be suitable for analyzing a larger data set, while other non-parametric algorithms, such as support vector regression (SVR), are more suitable to be used with small data set [30, 47] and in grasslands and shrubs AGB estimations [138]. However, regardless of the algorithm applied to the model (e.g., linear regression, RF, and ANN), independent variables seem to be more important to obtain accurate forest AGB estimations [30].
The predictive models are able to explore and rank the variables importance measure in the forest AGB estimation. Textural features from optical data (spectral data) and SAR (backscattering data), spectral vegetation indices, and, more recently, biophysical variables derived from Sentinel-2 (e.g., LAI - Leaf area index, FVC - Fractional vegetation cover, and FAPAR - fraction of photosynthetically active radiation) have been considered as the most important variables for forest AGB estimation [30, 47, 141, 142, 143, 144, 145]. Spectral bands produce predictive models with lower accuracy than using vegetation indices, transformed images and textural features [132]. Therefore, Forkuor et al. [44] showed that SWIR bands are important in predictive models of AGB estimation in semi-arid regions. In addition, the integration of variables (e.g., multispectral bands, transformed images, vegetation indices, and textural features) from optical and SAR sensors provide more accurate predictive models of forest AGB estimation [10, 52] than simple backscatter (SAR) and spectral (optical) bands [30, 47, 141, 142].
Vegetation indices are still important variables to estimate forest AGB as reported by several authors [59, 107, 134, 139, 140]. In last years, due to the Multi-Spectral Instrument aboard of the Sentinel-2 satellite, two relatively new vegetation indices, NDI45 and IRECI emerged (Table 2). Both new vegetation indices take advantage of the Sentinel-2 Red-edge bands (band 5 = 705 nm; band 6 = 740 nm; band 7 = 783 nm) to reduce the effects of saturation problem in high AGB density [44]. NDI45 is similar to NDVI but the original NIR band of 800 nm [84], is replaced by the new Red-edge band (band 5) and the Red band (band 4 of 665 nm) is kept [96]. On the other hand, IRECI uses the three available Red-edge bands of Sentinel-2 and put little emphasis in the red band to avoid saturation problem [97].
Transformed images, such as PCA, are also an important variable to face the saturation problem of optical sensor at low to intermediate biomass levels (between 60 and 150 Mg/ha) [146]. These images can also be used as input for textural images of optical and SAR sensors to prevent the saturation problem of high AGB density. For instance, textural variables showed to be more suitable to predict forest AGB estimation due to its ability to simplify complex cover structures, such as uneven-aged forests and different canopy structure then spectral bands [147, 148]. Also, textural bands from optical sensor images (e.g., sentinel-2, SPOT-6, and AVNIR) contributed to obtain accurate predictive models of forest AGB estimation than the original spectral bands [47, 132, 147, 149].
In addition, the greater interaction capacity of SAR-derived variables with the forest elements, such as branch, stem, and soil under the canopy [39, 65, 150], highlight their advantage over biophysical parameters (e.g., LAI, FVC, and FAPAR) to estimate forest AGB [44]. Hence, the importance of SAR long wavelengths (P-band), capable of providing accurate forest AGB estimations, will be harnessed in the BIOMASS mission to provide unprecedented information on the distribution of world’s forest AGB and its changes [17, 18]. This mission will help to build a sustainable global system of monitoring and quantification of biomass over time to help countries in managing forest resources and mitigating the impacts of climate change and land use changes.
From the analysis of several forestry AGB estimation studies, the integration of optical and radar data improves the information extraction process, taking advantage of the strengths of different image data. In this way, mixed pixel problems and data saturation is reduced. Further, Sentinel satellite family showed to be promising free satellites data to reach accurate forest AGB estimation models, including in regions with few or scarce AGB information.
Non-parametric models, such as RF, SVM, and ANN, have been replacing regression models due to their greatest ability to capture the heterogeneity of forest AGB than parametric models. Among the variety of machine learning techniques, RF algorithm showed to be one of the most used with ability to obtain better accuracy in forest AGB estimation, either using optical or SAR data.
The integration of different data sources RS-derived, that is, spectral bands, transformed images, vegetation indices, textural features, showed good correlation with forest AGB. VNIR bands are the most important to calculate most of vegetation indices. When using Sentinel-2 data, the available red-edge bands showed to reduce the effects of saturation problem in high AGB density.
PCA is a key variable to face the saturation problem of optical sensor of high AGB density and to be used as input data for textural features of optical and SAR sensors also to prevent the saturation problem of both sensors. Textural features, from both optical and SAR sensors, are among of the most suitable variables for forest AGB estimation due to their stronger relationships with AGB. SAR long wavelengths bands (L and P) showed to be very promising bands in studies of relatively high biomass density.
This work is funded by the National Funds through FCT - Foundation for Science and Technology under the Project UIDB/05183/2020 and by Programa Operativo de Cooperação Transfronteiriço Espanha-Portugal (POCTEP), and Programa INTERREG V A Espanha – Project IDERCEXA – Investigación, Desarrollo y Energías Renovables para nuevos modelos empresariales en Centro, Extremadura y Alentejo, 0330_IDERCEXA_4_E.
Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder characterized by skeletal muscle wasting that is resulted from mutations in the dystrophin gene [1]. The disease occurs at a frequency of about 1 in ~5000 newborn males, making it the most common severe neuromuscular disease in humans. Dystrophin is present in normal individuals from fetal life onwards in all skeletal, cardiac, and smooth muscles; the absence of dystrophin protein causes muscle weakness and protein degradation and ultimately causes cell death. Death usually occurs in the third decade of life as the result of respiratory or heart failure [2]. The precise diagnosis for DMD should contain a combination of genetic testing after muscle biopsy and clinical observation of muscle strength and function.
The main current medication so far is corticosteroids, which have been shown to increase muscle strength in many studies. Genetic therapy using mini-/microdystrophin vectors, suppression of premature termination codon, exon-skipping antisense oligonucleotides (AOs) which bind with RNA and exclude specific sites of RNA splicing producing a dystrophin that is smaller but functional, and such new emerging drugs are the pass to the new era towards DMD treatment. In the next section, we will review all available FDA-approved treatments and recent research trials aiming at ameliorating DMD symptoms.
Corticosteroids were the first line of treatment for DMD; it was first used by Drachman et al. in 1974 [3] when they had promising positive results in their study after using prednisone (anti-inflammatory glucocorticosteroid). Since then, many studies were carried out to test the therapeutic effect of such treatment since it was found to improve muscle performance.
Deflazacart (DFZ), an oxazolidine derivative of prednisone, was used by an Italian group [4] and other groups [5, 6, 7], and the drug demonstrated efficiency in disease treatment and preserved lung function. The exact mechanism of DFZ is not yet known; however, it might regulate some signaling cascades. It was found to activate calcineurin/NF-AT pathway [8]. Also, DFZ may act by decreasing necrosis and muscle inflammation and reducing the degree of muscle degeneration. It can also act through modulating dystrophin expression and inducing the myogenesis in addition to having positive effects on muscular tissue mass [9].
Despite the advantages of using steroids, they also had side effects like gaining weight, affecting bone mineral density, which leads to vertebral fractures and behavioral changes. Furthermore, high dosages are required to reach the target effect and to be active at the site inflammation. Also, the drug can be accumulated in other nontargeted areas [10, 11].
In one of their studies, Luhder et al. [12] tried to improve the therapeutic effect of the steroids through developing an 80 nm PEGylated nano-liposome that is conjugated with the steroid prodrug “methylprednisolone hemisuccinate.” The results of their study showed that such structure was selectively targeting the diaphragm in vivo (using mdx mouse model) when administered intravenously and the treatment reduced the infiltration with macrophages and serum levels of transforming growth factor beta. Most importantly, the study showed that long-term use of this formulation leads to enhanced mobility and increased muscle strength.
Exon skipping is considered as one of the mutation-based treatments for Duchenne muscular dystrophy [13]. In DMD, some deletions in specific exons lead to the disruption of the reading frame of the dystrophin protein, and consequently such deletions lead to the production of truncated product missing a huge part of the protein (usually missing the rod domain and C-terminal domain).
However, sometimes, deleting additional exons may restore the reading frame and lead to the production of dystrophin protein missing only a portion of the central rod domain while the C-terminal domain remains intact, and hence the protein product in this case is lacking specific regions, but it is semi-functional and can induce Becker-like symptoms instead of the complete loss of the muscular function [14].
The main idea of exon skipping is using the “antisense oligonucleotide” molecules to induce the skipping of a specific exon (other than the already mutated one) and prevent it from being translated to restore the reading frame. As an example, patients with exon 45 deletion could be treated through the skipping of an additional exon 44. Eteplirsen (Exondys51™) based on phosphorodiamidite morpholino oligomer (PMD) is an FDA-approved antisense treatment to skip exon 51 for patients with mutation ▲49–50 [15]. Also, drisapersen (based on 2′-O-methyl phosphorothioate; 2′-OMePS-modified AOs) is one of the AOs that are designed to treat DMD patients with mutations that can be ameliorated by exon 51 skipping; however it was not approved by the FDA [16, 17].
Various modifications can take place to the sugar of the oligonucleotide or to the backbone of the oligo. This could include phosphorodiamidate morpholino, locked nucleic acid (LNA), or peptide-conjugated oligo. Regarding the morpholinos, the oligonucleotide backbone is replaced with the morpholino backbone which makes the oligonucleotide nontoxic and has high affinity to RNA molecules. The locked nucleic acids are oligonucleotides that have a modified ribose sugar where the 2′ oxygen is connected with the 4′ carbon atom which creates a locked ribose ring. Also, the LNAs are nontoxic with superior affinity to complementary targeted RNA sequences [18].
The main problem in developing such treatments based on the skipping is that it will only fit a small group of patients (a mutation-specific AO should be developed for each group of patients and will not be suitable for other patients); also some patients have deletions in critical parts of the protein, and hence skipping of other exons will not have a therapeutic impact (Table 1).
Chemistry | Route of administration | The used model | Treatment strategy | Treatment effects | Reference |
---|---|---|---|---|---|
Phosphorodiamidate morpholino oligomers (Ex6A, Ex6B, Ex8A, and Ex8G) | Intravenous | Neonatal CXMDJ | Exon 6–9 skipping | Dystrophin restoration across skeletal muscles (14% of healthy levels) Reduction of fibrosis and/or necrosis area | [19] |
Phosphorodiamidate morpholino oligomer (NS-065/NCNP-01) | Endo-Porter reagent | Fibroblasts from patients with DMD involving deletion of exons 45–52 or exons 48–52 and injected with MYOD for myotube differentiation | Exon 53 skipping | Restored dystrophin protein levels in the cells | [20] |
Phosphorodiamidate morpholino oligomer | Intramuscular and intravenous | mdx52 mouse model | Exon 51 skipping | Only the protocol was mentioned | [21] |
Phosphorodiamidate morpholino oligomer (NS-065/NCNP-01) | Intravenous | Patients with DMD | Exon 53 skipping | Increased dystrophin/spectrin ratio in 7 of 10 patients in TA muscle biopsies | [22] |
Pip6a-PMO; PMOME23, sequence GGCCAAACCTCGGCTT-ACCTGAAAT | Intravenous | Cmah-/-mdx mice | Exon 23 skipping | Dystrophin restoration in the heart Reduction in myocardial fibrosis Reducing maximum pressure and arterial elastance | [23] |
Inhibitor of CDC2-like kinase 1 (named TG693) | Oral Lipofectamine reagent | Male Jcl:TCR mice Patient-derived myotubes | Exon 31 skipping | It induces exon skipping and restored dystrophin expression in patient-derived cells. And it modulated splicing in mouse skeletal muscle | [24] |
Morpholino AOs targeting DMD exon 51 | Endo-Porter transfection Intramuscular | Immortalized DMD muscle cells hDMD/Dmd null mice | Exon 51 skipping | The rescue of dystrophin protein expression | [25] |
Studies conducted on treatment of DMD using exon skipping (during 2017–2019).
The sole cause of DMD is the presence of mutation that adversely affects the DMD gene. So, in order to permanently fix such mutations and treat this condition, patients could be provided with muscle cells harboring the normal copy of DMD gene. Since it is hard to get mature muscle fibers from a normal individual to be used as a source of healthy muscle cells with normal DMD gene, also the availability of such source of cells will not guarantee the process of grafting in the patient’s muscles since it could be subjected to rejection by the body and can initiate an aggressive immune response. Cell reprogramming and genome editing techniques efficiently aid in solving this puzzling dilemma [25]. The process of cell reprogramming paved the road towards developing normal muscle fibers by starting with patient-specialized adult cells followed by inducing the production of induced pluripotent stem cells (iPSCs) (using the Nobel prize-winning technology of reprogramming using specific transcription factors like Oct4, Sox2, Klf4, and L-Myc) [26]. Also, some microRNAs have the potential to reprogram the adult cells efficiently (like miR-302b, miR-372) [27].
After the reprogramming and the production of stem cells, gene editing technologies should be used to correct the mutation of the gene. CRISPR/Cas 9 is now a leading technology that is presently considered as an avenue for DMD treatment; the RNA-guided DNA endonuclease system allows the correction of the DMD segment which is essential for dystrophin restoration [28, 29].
In order to conduct a gene editing experiment with CRISPR/Cas9 system, two important elements should be provided: guide RNA (gRNA) specific for the target gene and Cas9 nuclease (Sp. Cas9 (from Streptococcus pyogenes; 4.10 kb) or Sp. Cas9 (Staphylococcus aureus; 3.16 kb)) or Cj. Cas9 (Campylobacter jejuni; 2.95 kb) that can cleave DNA strands where the guide RNA is bound and in the presence of three- to five-nucleotide proto-spacer adjacent motif (PAM) sequence to be digested. Upon the binding of the gRNA, Cas9 can induce a double-strand break which is then repaired by the cell through the nonhomologous end joining, and this will initiate a repair mechanism in which nucleotides will be added or deleted at the cleaved site which can consequently restore the reading frame of the DMD gene to the normal ORF. In some cases, single (or several) gRNA molecule could be designed to target splicing sites which can lead to the skipping of specific exon leading to the production of functional proteins. Additionally, base editing mediated by CRISPR/Cas9 could be obtained through Cas9 enzymes lacking the nuclease activity, so it can induce only a single-strand break. Such enzymes can catalyze base editing (A:T to G:C) through having a cytidine deaminase activity [30].
Ousterout et al. in their study used another editing protocol (zinc finger nuclease) to delete exon 51 from the transcript from patient-derived myoblasts [31]. Also, Young et al. carried out CRISPR/Cas9 experiment utilizing a single pair of guide RNAs to delete exons 45–55 in iPSC, and such deletion leads to the expression of stable dystrophin protein with improved membrane stability in derived skeletal myotubes and cardiomyocytes [32]. Another study by Duchene et al. utilized a single guide RNA to produce a hybrid exon which led to the production of functional dystrophin protein with completely normal structure [33]. The main advantage of this reprogramming protocol is that it allows performing an autologous grafting of the muscle cells to patients.
For the expression of the specific gRNA molecules inside the muscle cells, adeno-associated virus (AAV) vectors will be used. Sometimes, the expression of the gRNAs can lead to off-target effect due to the incorrect binding with another similar DNA sequence inside the host cell. In order to avoid this damaging effect, AAV vectors expressing multiple gRNA molecules could be used.
After the completion of the gene editing process, the edited cells would be treated with myogenic factors to convert the edited stem cells again to myoblasts for the myogenic differentiation (Table 2).
Plasmids (source of Cas9 and guide RNAs) | Route of administration | The used model | Treatment strategy | Reference |
---|---|---|---|---|
Adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 A pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) | Intramuscular injection | DMDΔ52 pigs | Excision of exon 51 | [34] |
SaCas9 expression plasmid Two gRNA expression cassettes driven by the human U6 pol. III promoter (AAV8 and AAV9) | Locally in the TA muscles | C57BL/10ScSn-Dmdmdx/J | Excision of exon 23 | [35] |
pSpCas9 expression plasmid AAV TRISPR-sgRNA-CK8e-GFP plasmid contained three sgRNAs driven by the U6, H1, or 7SK promoter and green florescent protein (GFP) driven by the CK8e regulatory cassette | Transfection reagent Locally in the TA muscles | Human DMD-derived iPSCs ▲Exon 44 DMD mice | Excision of exons 43 and 45 | [36] |
Streptococcus pyogenes Cas9 Single guide RNA (sgRNA-51) (AAV9-Cas9 and AAV9-sgRNA-51) | Locally in the cranial tibialis muscles | ▲Exon 50 canine model | Excision of exon 51 | [37] |
spCas9 and crDMDint2.1 and int2.6 gRNAs | Transfection reagent (linear polyethylenimine derivative) | Immortalized myoblasts from DMD patient | Excision of duplicated exon 2 | [38] |
Lenti-V2-Ugi-nCas9-AIDx or Lenti-V2-AIDx-nSaCas9 (KKH)-Ugi (2.5 μg) and pCDNA3 Ugi | Transfection reagent (lipid-based) | ▲51-iPSCs of a male DMD patient | Excision of exon 50 | [39] |
CRISPR-Cas9 variant (D10A Cas9 nickase (nCas9) or catalytically deficient D10A/H840A Cas9 (dCas9) from S. pyogenes) and a deaminase protein from various sources sgRNA (gX20) under the control of the U6 promoter (pAAV-ITR-ABE-NT-sgRNA) | Micromanipulator | Mouse zygote from DMD knockout mouse | Base editing of exon 20 | [40] |
Plasmids containing regulatory cassettes for expression of Cas9 or gRNAs flanked by AAV serotype 2 inverted terminal repeats (ITRs) | Electroporation Intramuscular | Fibroblasts isolated from male mdx4cv mice Male mdx4cv mice | Excision of exons 52 and 53 | [41] |
pX601-AAV CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA (PX601) | Transfection reagent (lipid-based) | Myoblasts | Excision of exons 47 and 48 | [33] |
DMD iPSC | Excision of exons 44–55 | [32] | ||
The gRNA cassettes gI43, gI52, gI53, and gI54.2 (targeting different introns) controlled by human U6 RNA polymerase III promoter Plasmids AU53_pAd Shu.gI52.gI53.PGK.Cas9.SV40pA, plasmids pLV.gI52 and pLV.gI53 | Transduction (by gelatin) | DMD myoblasts Δ48–50 and Δ45–52 | Excision of exon 53 | [42] |
pAAV-ITR-CjCas9-sgRNA, pAAV2/9 encoding for AAV2rep and AAV9cap, and helper plasmid | Intramuscular in the TA muscle | Knockout mice | Excision of exon 23 | [43] |
pSpCas9(BB)-2A-GFP (PX458) | Transfection reagent | DMD hiPSCs, hiPSC-derived cardiac muscle cells | Excision of exon 51, introns 47, 50, 54 | [44] |
pSpCas9(BB)-2A-GFP (PX458) | Intramuscular in the TA muscle | Mice ▲50 | Excision of exon 51 | [45] |
Purified Cas9 protein and in vitro transcribed gRNA | Gold nanoparticles | Primary myoblasts C57BL/10ScSn-Dmdmdx/J (mdx) mice | Excision of exon 23 | [46] |
pSpCas9(BB)-2A-GFP (PX458) | Nucleofection | Induced pluripotent stem cells (iPSCs) | Deleting exons 3–9, 6–9, or 7–11 | [47] |
Nuclease-expressing plasmids (TALENs, left and right; CRISPR, Cas9 and sgRNA) | Electroporation | DMD fibroblasts were derived from a DMD patient lacking exon 44 | Excision of exon 45 | [48] |
Cas9 mixed with 44C1, 44C2, 45C2, and 45C3 gRNAs produced via in vitro transcription | Electroporation | hDMD (Tg(DMD)72Thoen/J, 018900), C57BL/10 mdx (001801), and mdxD2 (D1.B10-Dmdmdx/J, 013141) | Excision of exons 45–55 | [49] |
AdV-Cas9-RFP AdG-gRNA-Donor | Transfection reagent (lipid based) | Skeletal muscle cell culture derived from C57BL/10ScSn-Dmd mdx/J | Excision of exon 23 | [50] |
Cpf1 gRNAs targeting the human DMD or the mouse Dmd locus (subcloned into pLbCpf1-2A-GFP and pAsCpf1-2A-GFP) | Nucleofection | DMD iPSC cells | Excision of exon 51 | [51] |
Studies conducted on treatment of DMD using gene editing techniques (CRISPR/Cas9) (during 2017–2020).
Gene therapy is one of the most appealing techniques that are used to deliver a normal copy of the DMD gene to express the fully functional dystrophin protein. This method implies injecting the patients with plasmids carrying normal dystrophin cDNA (~12 kb).
In 2002, the first phase 1 trial of DMD gene therapy took place using full-length dystrophin [52]. After that, adeno-associated viral vectors carrying mini forms of dystrophin cDNA were used for gene therapy, and this was better regarding the packaging size of the plasmids, and it is much easier to transfer/deliver mini forms of DMD gene [53, 54].
However, such therapeutic approach faced another problem which is the distribution of the plasmids across all affected muscular tissue that is spreading all over the body, and that is why microdystrophin plasmids and systemic AAV delivery were developed and improved to solve such problem. Evidence from many trials using animal models revealed that gene therapy can lead to long-term expression of functional protein [55, 56, 57].
In 2017, Le Guiner et al. studied the effect of the delivery of rAAV2/8 vector expressing a canine microdystrophin (cMD1) in golden retriever muscular dystrophy (GRMD) dogs in the absence of immunosuppression. Such treatment affected the deterioration of the muscular activity, and the gene expression was maintained over a long period [56]. Recently in 2020, Genthon and Sarepta contracted Yposkesi for manufacturing the AAV microdystrophin vector on a large scale.
As previously mentioned, the absence of dystrophin is the main cause of DMD disease and the aggressive symptoms including muscle weakness and degeneration of muscle fibers. Such defect in the DMD gene can be edited using gene editing technology; however such technology can lead to off-target mutations which consequently can have damaging effects, and that is why more therapies had to be developed to address the disadvantages of such techniques.
Chimeric cell therapy is an alternative therapeutic approach that is usually done through the fusion of normal myoblasts and dystrophin-deficient myoblasts using polyethylene glycol (PEG). The success of this process could be tested using immunophenotyping (flow cytometry) and dystrophin immunostaining. This fusion will be followed by transplantation of chimeric cells in the defected muscle. The chimeric cells always show behavior like the donor cells regarding dystrophin expression and myogenic differentiation, and this dramatically improves the muscle function after being transplanted [58].
Cardiosphere-derived cells are a type of cells that are retrieved from cardiac explants that can be cultured in vitro. Such cells have anti-inflammatory, antioxidant, and antibiotic activities. Several studies tested the ability of CDCs to alter the pathophysiology of DMD after the injection of these cells directly into the cardiac muscle.
Recently it was found that using CDCs greatly enhanced the phenotypic status of cardiac and skeletal muscles. The therapeutic effects of CDCs are usually attributed to the secretion of specific exosomes carrying specific genetic material to distal cells to exert its biological effect. Such CDCs along with their secreted exosomes can be injected intravenously, and it was found that they can greatly enhance the skeletal and cardiac muscle functions and boost the muscle generation process [59, 60].
In some of the mutations affecting the DMD gene, a premature stop codon is produced that can significantly disturb the reading frame and gives a truncated abnormal protein that cannot maintain the structural and functional properties of the muscle fibers.
A class of antibiotics called aminoglycosides was found to bind to rRNA at their decoding sites, preventing the stop codons from being read by binding to the A site (acceptor site) in the ribosomes and forcing the cell to prevent reading the stop codon, hence leading to the production of fully functional proteins.
PTC124 (ataluren; trade name, Translarna™) is one of the drugs with the read-through properties that are used for the treatment of DMD. Clinical trials showed that this drug when administered orally induced the expression of the dystrophin protein.
However, this treatment can only be used in ~15% of the cases who have a stop signal resulted from point mutation in the DMD gene. Also, it must be administered in increasing doses; beside it has many side effects such as renal toxicity. That is why there is a need to develop other alternatives with other structures to be safer with chronic usage.
Recently, DMD symptoms were found to be managed after the administration of utrophin protein expression enhancers (utrophin is a dystrophin homolog; 395 KDa in size) to DMD patients delays the need of wheelchair and efficiently substitutes non-functional dystrophin.
Like dystrophin, the utrophin is present in the sarcolemma in the first developmental stages, and then replacement with dystrophin took place during muscular maturation. However, utrophin was found to be present in the myotendinous junction in adults. Interestingly, the expression of utrophin becomes elevated as a normal repair mechanism to compensate the absence of functional dystrophin in regenerated muscles.
SMTC-1100 is one of the chemical molecules that showed a great potential to increase the expression of DMD transcript and protein as well. This drug can be administered orally, if it was found to be safe and well tolerated in volunteers. However further studies are still required to detect if high dosage of this drug is safe or not.
Recently, ASA or adenylo-succinic acid improved the status of the TA muscles in mdx mice after administration of this compound in the drinking water. This molecule regulated the expression of the utrophin protein and hence greatly reduced the damaged area [61].
Another study group designed AAV vector carrying mini forms of the utrophin protein (μUtro). Their results showed that expression of this functional copy of utrophin protein (dystrophin analogue) after administration of the utrophin vector in DMD animal models completely reduced the muscle necrosis and regeneration [62].
Many medications have been used for DMD treatment and for preventing further deterioration of the cases. Corticosteroids were the first line of effective therapy of DMD; however, it does not modify the genetic mutations of the gene and does not affect the expression levels of dystrophin protein. Consequently, other treatments were developed including read-through stop codon, gene therapies, and exon skipping AOs which modulate and upregulate the levels of functional dystrophin transcript and protein in the muscles. Genome editing technology is also a powerful tool that can treat DMD permanently through the correction of the mutated sequence of DMD gene through the administration of sequence-specific guide RNA strands to bind selectively in the sequence to be edited. Also upregulating utrophin can help in the management of the cases. In addition, dystrophin-expressing chimeric cells and cardiosphere-derived cells are two emerging techniques that have the potential to treat DMD. Other medications will be developed to treat all DMD patients with different mutations with minimum side effects and maximum improvement in the status of the muscular system.
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