Enzyme and Microbial Technology 172 (2024) 110348

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Enzyme and Microbial Technology

journal homepage: www.elsevier.com/locate/enzmictec

Chondroitinase as a therapeutic enzyme: Prospects and challenges

Asma Kheirollahi a, Solmaz Sadeghi b, Shirin Orandi c, Kiana Moayedi c, Khosro Khajeh d,
Mehdi Khoobi e, f, Abolfazl Golestani c, *
a
Department of Comparative Biosciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
b
Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
c
Department of Clinical Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
d
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-154, Iran
e
Department of Radiopharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
f
Department of Pharmaceutical Biomaterials and Medical Biomaterials Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: The chondroitinases (Chase) are bacterial lyases that specifically digest chondroitin sulfate and/or dermatan
Chondroitinase sulfate glycosaminoglycans via a β-elimination reaction and generate unsaturated disaccharides. In recent de­
Combinational therapy cades, these enzymes have attracted the attention of many researchers due to their potential applications in
Drug delivery
various aspects of medicine from the treatment of spinal cord injury to use as an analytical tool. In spite of this
Enzyme engineering
Spinal cord injury
diverse spectrum, the application of Chase is faced with several limitations and challenges such as thermal
instability and lack of a suitable delivery system. In the current review, we address potential therapeutic ap­
plications of Chase with emphasis on the challenges ahead. Then, we summarize the latest achievements to
overcome the problems by considering the studies carried out in the field of enzyme engineering, drug delivery,
and combination-based therapy.

1. Introduction
For the first time, bacterial degradation of chondroitin sulfate (ChS)
was described by Neuberg and Rubin as early as 1914 [1], and in the
following, it was found that cell extract of Pseudomonas fluorescens,
Pseudomonas aeruginosa, and Proteus vulgaris break down ChS into a
product having reducing power and sulfuric acid [2]. Among glycos­
aminoglycans (GAGs) lyase-producing bacteria, P. vulagris was the first
well-known organism to produce chondroitinase (Chase) [3]. Yamagata
et al. purified enzymes from the cells of P. vulgaris NCTC 4636 and Fla­
vobacterium heparinum ATCC 13125 [4]. It is indicated that P. vulgaris
produces two distinct Chase enzymes (PvCS ABC I and II), which de­
grades ChS-A, ChS-B, ChS-C and hyaluronic acid, and F. heparinum
(currently known as Pedobacter heparinus) produces two Chases with
different substrate specificity (FlavoAC and FlavoB) (Fig. 1) [4–6].
Chase can cleave chondroitin sulfate by a β-elimination reaction, which
results in the formation of unsaturated oligosaccharides. This mecha­
nism is based on proton acceptance and donation. Initially, a double
bond is formed between C-4 and C-5 as a result of the elimination of the
C-5 proton from the uronic acid moiety. This double bond formation is
the basis for the enzyme activity assay at 232 nm. After stabilization of
the carbanion intermediate the anomeric oxygen protonates and leads to

Abbreviations: AAV, adeno-associated virus; ADSCs, adipose-derived mesenchymal stem cells; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast
growth factor; BMSCs, bone marrow stromal cells; Chase, chondroitinases; ChS, chondroitin sulfate; CNS, central nervous system; CNTF, ciliary neurotrophic factor;
CSPGs, chondroitin sulfate glycosaminoglycans; db-cAMP, dibutyryl-cyclic adenosine monophosphate; DRG, dorsal root ganglia; ECM, extracellular matrix; EGF,
epidermal growth factor; ENSCs, enteric neural stem cells; GAP43, growth associated protein 43; FGF2, fibroblast growth factor 2; GDNF, glial cell-derived neu­
rotrophic factor; hUSCs, human urine stem cells; hADSCs, human adipose-derived stem cells; IGF, insulin-like growth factor; KLF7, Krüppel-like Factor 7; LV,
lentiviral virus; MSCs, mesenchymal stromal cells; EP1–40, Nogo-66 antagonist peptide; NESCs, neuroepithelial stem cells; NGF, nerve growth factor; NPs, Nano­
particles; NSCs, neural stem cells; NT-3, Neurotrophin-3; OECs, olfactory ensheathing cells; oNPCs, directly reprogrammed human NPCs biased toward an oligo­
dendrogenic fate; OPCs, Oligodendrocyte progenitor cells; OV, oncolytic virus; PDGF, platelet-derived growth factor; PEG, polyethylene glycol; PLGA, Poly(lactic-co-
glycolic acid); PLL, poly L-lysine; pMNs, ESC-derived progenitor motor neurons; PNGs, peripheral nerve grafts; PPN, pre-degenerated peripheral nerve; PVD, pos­
terior vitreous detachment; RHEB, Ras homolog enriched in brain; SCI, spinal cord injury; SH3, Src homology 3; TGFβ, transforming growth factor beta.
* Correspondence to: Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, P.O. Box: 14155-6447, Tehran, Iran.
E-mail address: golsetan@tums.ac.ir (A. Golestani).

https://doi.org/10.1016/j.enzmictec.2023.110348
Received 22 May 2023; Received in revised form 28 September 2023; Accepted 19 October 2023
Available online 21 October 2023
0141-0229/© 2023 Elsevier Inc. All rights reserved.
A. Kheirollahi et al.
the breakage of the glycosidic bond [7]. Thses enzymes can be classified
into two distinct categories based on their degradation patterns:
endo-lyases and exo-lyases. Endo-lyases initially fragment GAG chains
into larger oligosaccharides and eventually into disaccharides, following
a random cleavage pattern. Conversely, exo-lyases systematically
remove disaccharide residues from the terminal end of the GAG chain,
without generating larger oligosaccharides in the course of their enzy­
matic action [8]. Nowadays, P. vulgaris, F. heparinum, and Arthrobacter
aurescencs (currently known as Paenarthrobacter aurescens) are used for
commercial production of Chase [4,5,9,10]. Recently, an extracellular
Chase ABC with the highest activity at 42 ◦ C is purified form Acineto­
bacter sp.C26. In spite of various Chase-producing bacteria strains, it is of
paramount importance to identify new microbial sources for obtaining
an enzyme with improved catalytic activity and structural stability [11].
Chase could have various medical and/or therapeutic applications
through the degradation of chondroitin sulfate glycosaminoglycans
(CSPGs) which increment in their level is related to various weakening
circumstances [12–14]. Nevertheless, the Chase application in medicine
is faced with severe challenges. The current review addresses the ther­
apeutic applications of Chase and the challenges ahead in this way and
finally summarizes the latest findings to overcome the problems
regarding enzyme engineering, drug delivery, and combination-based
therapy.
2. The structure of chondroitinase
GAG lyases are not found in vertebrates [15] and are divided into
three classes by substrate specificity: heparinases, chondroitinases, and
hyaluronidases [16]. Until now, three folds ( (α/α)5-toroid, right-handed
β-helix, and β-sandwich) have been recognized in these enzymes [15].
Among several types of Chases, Chase ABC is the most important enzyme
for therapeutic use and research. The following sections introduce the
structure of Chases, briefly.
Chondroitinase AC. As shown in Fig. 1, Chase AC is mainly pro­
duced by A. aurescens and F. heparinum bacteria [5,9]. Structurally,
FlavoAC is a two-domain molecule consisting of several α-helices in the
N-terminal domain and four β-sheets in the C-terminal domain (Fig. 2A)
[17]. The N-terminal domain, which includes the catalytic site and a
significant portion of the substrate-binding site, has an incomplete
double-layered (α/α)5-toroid fold [17].
Fig. 1. Microbial sources of chondroitin lyase [4–6,9,32,252,253].
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Enzyme and Microbial Technology 172 (2024) 110348
Chondroitinase B. Chase B is produced by F. heparinum bacteria
(Fig. 1) [5]. The Cygler laboratory determined the structure of this lyase
(Fig. 2B) [18]. Based on the crystal structure, Chase B is a member of the
β-helix family and has different folds from FlavoAC [18]. Moreover,
Chase B requires calcium for its activity and the crystal structure un­
ravels its calcium-dependent catalytic machinery [19].
Chondroitinase ABC. The bacterium Proteus vulgaris generates two
Chases with wide substrate specificity, named Chase ABC I (PvCS ABC I)
and Chase ABC II (PvCS ABC II) [6]. The catalytic mechanisms of these
enzymes are different. PvCS ABC I is an endolytic enzyme with 997
residues; whereas isoform PvCS ABC II has 990 residues and shows
exolytic activity [6]. While the crystal structure of PvCS ABC II has not
yet been determined, the PvCS ABC I structure was crystallized and
characterized by the Cygler laboratory (Fig. 2C) [16,20]. PvCS ABC I is a
110 kD protein which is comprised of three domains with linear fashion
arrangement [16]. The N-terminal domain of PvCS ABC I (residues
25–234) possesses a jellyroll fold and shows the highest structural
flexibility (Fig. 2D) [16]. The central domain of PvCS ABCI consists of
residues 235–617 with an incomplete (α/α)5 toroid topology (Fig. 2D)
[16]. Mutagenesis studies have revealed that four structurally conserved
amino acid residues –including His-501, Tyr-508, Arg-560, and Glu-653-
have a critical role in Chase ABC I catalysis [7,16,21]. The C-terminal
domain of PvCS ABCI includes 618–1021 residues and is formed of the
stacked β-sheets (Fig. 2D) [16].
3. Production of chondroitinase
Various bacterial strains, as shown in Fig. 1, are known for their
ability to produce Chase. Nevertheless, P. vulgaris, F. heparinum, and
A. aurescencs have gained prominence as commonly utilized bacterial
sources in the commercial production of Chase [4,5,9,10]. Researchers
have optimized fermentation conditions to enhance Chase yield, by
controlling pH, optimizing induction media and adjusting temperature
[22–24]. Besides bacterial sources, there have been reports of fungal
Chase production [25]. In this context, Kasinathan et al. optimized the
cultural conditions for the production of chondroitinase by Aspergillus
niger and documented a maximum chondroitinase yield of 22.5 U/ml
[26]. Challenges in Chase production include downstream processing
and purification to obtain highly active and pure enzymes for thera­
peutic use.
A. Kheirollahi et al. Enzyme and Microbial Technology 172 (2024) 110348

Fig. 2. 3D structure of chondroitinases. Protein structures of FlavoAC (A), FlavoB (B), and PvulABC I (C) are shown in cartoon representation and colored based on
the secondary structure elements. (D) Distinct domains of chondroitinase PvulABC I are illustrated in different colors, also, some mutated residues (in N- and C-
terminal domains) and involved residues in catalysis (in catalytic domain) are labeled. These residues are colored in red and displayed in stick representation.
Visualization is done by PyMOL software (PDB ID: 1CB8, 1DBG and 1HN0). Refer to the text for the details.

For the research objectives and commercial-scale production of 28]. The production process of recombinant Chase typically involves
Chase, genetic engineering techniques have been strategically employed several key steps, as depicted in Fig. 3 for the production of Chase ABC I
to introduce Chase genes into high-efficiency expression systems, facil­ from P. vulgaris. In the majority of studies, Escherichia coli has been
itating the production of recombinant Chase on a substantial scale [27, chosen as the preferred host for the production of recombinant Chase,

3
A. Kheirollahi et al.
Fig. 3. Flowchart diagram of production process of recombinant chondroitinase ABC I from P. vulgaris in E.coli [27,122].
due to its well-understood genetics and ease of manipulation [27–30]. In
this context, E. coli strains DH5α and BL21 (DE3) have been commonly
employed as appropriate strains for ligation reactions and enzyme
expression, respectively [27,28]. To date, cloning and recombinant
expression of Chase from P. vulgaris, F. heparinum, Bacteroides stercoris
and A. aurescencs have been documented [21,28–31]. In 2001, Pojasek
et al. reported the cloning of Chase AC and B from F. heparinum in
pET15b and pCRT7/NT vectors. To facilitate the expression of Chase AC
and Chase B, an inducible T7 promoter was utilized in both the pET15b
and pCRT7/NT expression systems, alongside the incorporation of an
N-terminal His6 tag to enhance purification efficiency. Their results
indicated that Chase AC, when produced from pET15b, achieved the
highest yield, yielding 24.4 mg of protein and a total activity of 8820 U
per liter of culture. In contrast, Chase B expressed in the pCRT7/NT
vector produced a higher amount of protein compared to the pET15b
clone, with a yield of 10.4 mg of enzyme and a total activity of 880 U
[28]. In the following, Prabhakar et al. cloned Chase ABC I from
P. vulgaris in pET28a vector and reported the expression of the enzyme
with an N-terminal His6 tag in E.coli. They indicated that the Chase ABC
I purification process resulted in the production of more than 35 mg of
protein from a 500 ml culture [27].
The production of Chase from bacteria is affected by several key
factors. The choice of the bacterial strain is crucial, as different strains
may have varying levels of Chase production capability [32]. Opti­
mizing culture conditions, such as temperature, pH, aeration, and
agitation, is essential for maximizing enzyme yield [33]. Additionally,
the composition of the growth medium plays a significant role, with
carbon and nitrogen sources, as well as trace elements, affecting Chase
production [33]. The optimized cultural conditions for bacterial cells
containing a vector carrying the gene encoding Chase have been docu­
mented, with minor variations observed among different studies [27,28,
34]. Recently, Lu et al. conducted an optimization of the culture con­
ditions for the expression of Chase ABC I using response surface meth­
odology. Their findings revealed that the expression level achieved was
1.65 times higher than that observed in the shake flask setup. Addi­
tionally, they incorporated a signal peptide at the N-terminal of the
Chase ABC I gene and indicated that this signal peptide facilitates the
folding of Chase ABC I and prevents its formation as inclusion bodies
[35]. To date, the use of various inducible promoters (e.g. T7 and AOX1)
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Enzyme and Microbial Technology 172 (2024) 110348
and genetic modifications have been examined to enhance enzyme
expression [31,35,36]. Effectively regulating fermentation parameters
and scaling up production processes are vital for achieving elevated
yields of Chase enzyme. However, challenges in maintaining enzyme
stability and preventing proteolysis during production need to be
addressed to ensure the production of active and therapeutic-grade
Chase.
4. Medical applications of chondroitinase
In spite of important biological functions of CSPGs, increased levels
of these molecules are related to various weakening circumstances.
Therefore, chondroitinase can have direct medical applications in states
where there is an up-regulation in the amount of chondroitin sulfates,
such as spinal cord injury (SCI) [12], cancer [37], vitreous attachment
[38] and keloid scars [39] (Fig. 4). Among various types of chon­
droitinases, Chase ABC is the most efficient one due to its broad sub­
strate spectrum, and over the last decade, numerous experimental
studies confirmed that this enzyme could be a promising drug candidate
for the treatment of various pathological conditions. In this context,
researches have focused on optimizing the beneficial effects of the
enzyme in vivo.
4.1. Chondroitinase and the nervous system
Several studies have indicated that Chase ABC improves nerve
regeneration in the central nervous system (CNS) [12,40]. Following
spinal cord injuries, regeneration of the axons would be inhibited in
adult mammals due to a glial scar formation at the injury site [41]. The
glial scar consists of extracellular matrix (ECM) molecules and is espe­
cially rich in CSPGs [41]. In vitro and in vivo studies showed that CSPGs
inhibit axonal growth and regeneration [42–45]. Numerous studies have
demonstrated that Chase ABC which catalyzes the digestion of CSPGs,
can reverse the repressive activity of CSPGs in vitro [46–49]. Moreover,
in a Lamprey model of SCI, Chase ABC caused not only digestion of
CSPGs in the spinal cord but also, led to a significant reduction in
retrograde apoptosis by reducing the number of caspase-3 positive
neurons. On the other hand, Chase ABC treatment showed a great
reduction in PTPσ mRNA expression, a feasible receptor of CSPGs.
A. Kheirollahi et al.
Fig. 4. Potential applications of chondroitinase.
Furthermore, after enzyme therapy, Akt activation, which is known to
be a mechanism to promote axonal regeneration, was increased, sug­
gesting the possible role of Chase ABC in apoptosis inhibition through
the reduction of PTPσ expression and Akt activation [50] Lemon et al.
have reported the reducing level of CSPGs after SCI by delivery of Chase
ABC in vivo [51]. A significant breakthrough came by Bradbury et al.,
and they have observed a recovered locomotor function in the rat
models of SCI after treating them with Chase [12]. In the following,
Tester et al. proved the therapeutic efficacy of Chase in higher animals
[52]. They demonstrated that Chase can recover the basic and skilled
locomotor function in hemisected cats [52]. Critically, the robustness of
efficacy of Chase has been demonstrated in a rat model of chronic SCI
[53], so potentially, numerous patients may make a profit from this
treatment.
Although ischemic stroke induces sustained sensorimotor impair­
ments, several studies indicate that Chase may be a valuable treatment
to reduce stroke and brain injury inability [54–57]. It has been found
that Chase ABC improves the recovery motor function of the affected
limb after stroke and induces synaptic plasticity in rats [54,56,57].
Based on these findings, Chase ABC can be considered as a novel ther­
apeutic candidate in the acute and/or chronic phase of ischemic injury
for aged individuals [54]. The effects of Chase ABC on the plasticity of
the axon, neuronal connections, cell adhesion, diffusion of the growth
factor, and inflammation have been investigated in different studies
[58–61], even though the mechanisms underlying Chase ABC-induced
improvement following stroke is not exactly defined. However, it
seems that the mechanisms of Chase ABC-induced improvement in the
spinal cord and brain injuries may vary [62]. In addition to increasing
axonal regeneration in the brain, as shown in the mice model of trau­
matic brain injury, Chase ABC can decrease post-traumatic brain edema
[63].
Treatment of amblyopia, also called lazy eye, is another possible
therapeutic application of Chase. Amblyopia is decreased sight in an eye
due to the loss of visual cortex plasticity [64]. To date, complete re­
covery of vision in adults with cortical visual impairment is therapeu­
tically infeasible. However, Pizzorusso et al. induced plasticity of the
visual cortex in adult rats by Chase ABC and attributed weak visual
cortex plasticity to the presence of CSPGs [40,65]. In contrast, a study
the effect of Chase ABC on plasticity in the visual cortex of an adult cat
showed that the application of the enzyme was not associated with
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Enzyme and Microbial Technology 172 (2024) 110348
significant functional recovery of the deprived eye [66].
Apart from several studies about Chase ABC’s well-known role in
spinal cord injury, its ability to create a favorable environment for
axonal regeneration has also been investigated in other scenarios. After
glaucomatous degeneration, an early feature of neuronal damage due to
Glaucoma, CSPGs restrict retinal ganglion cell regrowth by their ability
to inhibit neuronal plasticity. With the use of rat models of inducible
glaucoma, R. Tribble et al. observed that although injection of Chase
ABC couldn’t affect intraocular pressure (IOP) associated with injury, it
showed significant preservation of dendrites in injured eyes [67].
Given the beneficial effect of Chase on neural growth, it seems that
the enzyme has the potential for wider therapeutic application. In
addition to the CNS-related pathological states, using Chase has shown
improved outcomes following peripheral nerve injury [68,69]. More­
over, it has been found that CSPGs, which are present in the infarct after
ischemia-reperfusion, inhibit sympathetic regeneration axon outgrowth
in vitro [70]. Thus, Chase ABC can be used after myocardial infarction to
stimulate cardiac sympathetic nerve regeneration [70].
Also, the neuroprotective effect of Chase ABC treatment in partial 6-
hydroxydopamine (6-OHDA) lesion mouse models of Parkinson’s dis­
ease were reported by R. Fletcher et al. because of the enzyme’s ability
to preserve both substantia nigra pars compacta (SNc) cells and dopa­
minergic fibers. However, Chase ABC treatment in mice bearing full 6-
OHDA lesions failed to show such results. Also, despite the protective
function of Chase ABC treatment in partial 6-OHDA lesions, it didn’t
show any behavioral improvement in these animals [71].
4.2. Chondroitinase and cancer
Changes in proteoglycan levels are associated with different patho­
logical circumstances, including cancer [72]. Recent findings suggest
that CSPGs have a significant role in tumor development, angiogenesis,
and metastasis by regulating cell-ECM, cell-cell, and cell-soluble factors
interactions in cancer/host tissues [73–77]. Given the diverse functions
of cell surface CSPGs in controlling cancer growth, migration, and in­
vasion, targeting these molecules can be considered as a therapeutic
strategy. In this context, the efficacy of targeting cell surface PGs for
inhibiting tumor growth and metastasis has been evaluated in some
studies [14,37]. In 1972, Takeuchi et al. inhibited tumor growth through
targeting cell surface CS by treating them with Chase AC in vitro and in
vivo [37]. After that, many researches demonstrated that hindering the
production of CSPGs with β-D-xyloside, and/or enzymatically digesting
them with Chase ABC, reduced the metastatic potency of tumor cells
[78–80]. In 2001, Denholm et al. showed that Chase suppresses invasion
and proliferation in melanoma cells [14]. Moreover, it has been
demonstrated that enzymatic removal of tumor-cell surface CS-GAGs by
Chase ABC significantly reduces lung metastasis in the 4T1 murine
mammary tumor model [81]. Since the nature of a CSPG varies with the
type of tumor [82–84], the efficacy of various kinds of Chases is related
to the nature of the ECM surrounding the tumor. It seems that the
relative amount of GAGs in various kinds of cells and their linkage to
different membrane receptors probably determines the level of CSPGs’
influence on many cellular activities. Thus, Chase possibly decreases cell
proliferation by an alteration in binding between some ligands (e.g.
hepatocyte growth factor, platelet factor 4, interferon-γ and so on) and
their corresponding receptors [85–89]. Although Chase is considered to
inhibit tumor growth in a variety of ways, activation of caspase-3 and
subsequent induction of apoptosis is expected to be one of the key action
mechanisms of Chase in the treatment of cancer [14]. Despite promising
results, the use of Chase in cancer treatment remains controversial due
to some conflicting results. In contrast to the mentioned studies, in a
study of breast cancer in Balb/c mice, intra-tumor injection of Chase
ABC caused higher lung metastases and the development of secondary
tumors [90].
Further application of Chase in cancer therapy is its usage as an
adjuvant. The efficiency of using an oncolytic virus (OV) in the
A. Kheirollahi et al.
treatment of cancer is limited owing to the failure of the viruses to
efficiently diffuse and infect tumor cells in the presence of tumoral
CSPGs [91]. Dmitrieva et al. demonstrated that removal of the glioma
ECM by using a Chase-expressing oncolytic virus enhances virus spread
and anti-cancer effectiveness [92]. Recently, Jaime-Ramirez et al. pro­
duced a humanized Chase enzyme-expressing OV and revealed that the
viral susceptibility of glioma cells and subsequently their sensitivity to
temozolomide was improved [93]. The studies show that the use of
Chase-expressing OV in the treatment of gliomas is safer than other
adjuvants [92,93].
In addition to the therapeutic application of Chase in cancer, this
enzyme can serve as a laboratory diagnostic tool to precisely detect CS.
For example, a significant increase in non-sulfated disaccharide levels in
the urine of patients with cancer of the head and neck has been reported
so that this elevated level reduces after the removal of cancer [94]. Thus,
analysis of the presence of CS in urine samples by using various types of
Chases may be useful in the diagnosis and follow-up of cancer.
Several studies have shown CSPGs’ role in the development of cancer
since they play a significant role in metastasis and angiogenesis, there­
fore Chase ABC seems to be a promising approach in the treatment of
cancerous cells [10]. Additionally, it is believed that the extracellular
matrix’s (ECM) structural components such as proteoglycans create a
barrier for the distribution of large therapeutic molecules. Having this in
mind, a study by Dmitrieva et al. [95] used Chase ABC to digest CS-GAGs
in cultured glioma cells to improve the oncolytic virus (OV) ability to the
destruction of cancerous cells. The results of this study showed that
Chase ABC-mediated digestion of ECM improved OV diffusion in glioma
grown in brain slices. Then, they administered OV expressing Chase ABC
in intracranial xenografts and this resulted in longer survival of animals
without affecting Glioma cell invasion and migration. Another study
demonstrated the effect of temozolomide combined with a mutant hu­
manized version (Chase M) in order to improve the chemotherapeutic
sensitivity of cultured glioma cells. This combinational treatment
increased glioma cell death. Additionally, using Intracellular flow
cytometric analysis, it was revealed that this combinational treatment
resulted in a significant reduction in AKT phosphorylation, a
pro-survival protein, in cultured cells [93]. On the contrary, in a study of
breast cancer in Balb/c mice, intra-tumor injection of Chase ABC caused
higher lung metastases and the development of secondary tumors.
Therefore, it can be concluded that chondroitin sulfate might act as an
inhibitor for cancerous cells spread and metastasis [90].
4.3. Enzymatic vitrectomy, enzymatic nucleolysis and keloid scar
In a few people, the vitreous cortex is connected to the internal
limiting lamina of the inner retina [96]. This adhesion between the
vitreous and the retina participates in certain pathological conditions,
like diabetic retinopathy [96]. Vitrectomy which is a surgical technique
of removing the vitreous cortex entirely from the retina, decreases
macular edema and improves visual acuity in the eyes of diabetic pa­
tients [97]. In this context, many pharmacologic agents including en­
zymes e.g. hyaluronidase, plasmin, nattokinase, and chondroitinase
have been investigated to induce a posterior vitreous detachment (PVD)
without harming the retina before vitrectomy [97,98]. Researchers
showed that intravitreal injection of Chase in monkeys and human
donor eyes induced the separation of the vitreous from the retina [38,
97]. However, some studies showed that the efficacy of Chase in vitre­
oretinal separation is lower than the other enzymes. For example, Her­
mel and Schrage demonstrated that Chase is not able to induce PVD in
pig eyes when compared to plasmin [99].
Enzymatic nucleolysis is a non-surgical procedure for treating her­
niated disk of the lumbar spine that involves removing the gelatinous
cushioning material in an intervertebral disk by the injection of an
enzyme [100]. In 1990, Chase ABC was applied therapeutically to treat
invertebral disc protrusion [101]. Investigations showed that using of
Chase in nucleolysis is more secure than the broadly examined
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Enzyme and Microbial Technology 172 (2024) 110348
chymopapain [13]. In this context, studies in canines [102], goats [103],
monkeys [104], and sheep [105] confirmed that Chase would be a good
candidate for nucleolysis.
Since CSPGs are up-regulated in keloid matrixes [106], Chase may be
helpful to treat keloid scars as shown in mice grafted with keloid im­
plants and ex vivo models [39,107]. However, further investigations are
needed for evaluating the effect of Chase alone or in combination with
other enzymes in vivo.
5. Challenges in the medical application of chondroitinase
Although the beneficial effects of Chase in treating the above-
mentioned pathological conditions have been proved in experimental
and/or animal models, clinical translation remains challenging, partic­
ularly in the case of SCI treatment (Fig. 5). For example, the formation of
CSPGs continues over weeks after the SCI [108,109], whereas Chase
ABC quickly loses its enzymatic activity at body temperature [110]; So
that its activity remarkably diminished after 3 days and completely lost
by 5 days at 37 ◦ C [110]. Therefore, thermal instability of currently
available Chase and also diffusion of the enzyme far from the injury site,
necessitate frequent intrathecal injection of the enzyme [111,112].
However, repeated intrathecal injection is highly invasive, painful, and
infection-prone, and increases the risk of physical injury [111,113].
Hence, finding a thermostable enzyme and/or a delivery procedure that
will ensure continuous catalytic activity of the enzyme at the injury site
over long time intervals are big challenges of the therapeutic application
of Chase. In this regard, the delivery methods tested in animal models,
like local infusion pumps and intrathecal catheters, would be hard to
translate into the human, because of the need for highly skilled physi­
cians and enhanced the risk of infection [114]. In general, while the
therapeutic power of Chase is promising, further experimental in­
vestigations are needed to overcome the challenges facing the applica­
tion of the enzyme in vivo (Fig. 5) Researchers attempt to overcome
these obstacles through improving thermal stability of Chase, enzyme
delivery by viral vectors, delivery within biomaterials, and generation of
Chase-secreting cells.
Although chronic delivery of Chase carries risks of immunogenicity,
no study to date has addressed this issue. In addition to thermal insta­
bility, delivery and immunogenicity of chondroitinase, lack of speci­
ficity is another potential issue facing the use of Chase ABC in SCI
treatment, because it digests both inhibitory and growth-promoting
proteoglycans [115]. In this regard, delayed injection of growth fac­
tors after Chase ABC treatment and co-administration of Chase ABC with
cells that often express and release growth-promoting factors to ECM
may circumvent this problem. Recently, several studies have indicated
that the delivery of Chase ABC in combination with other approaches is
significantly more efficient. The following sections discuss the applied
strategies to make Chase ABC as a more impressive therapeutic agent
-particularly for SCI- by using protein engineering approaches, drug
delivery methods, and combinational therapies (Fig. 5).
5.1. Thermal stabilization
Several approaches have been widely used to improve the structural
stability of enzymes, such as enzyme engineering, immobilization,
chemical modifications, and utilization of additives [116,117].
Site-directed mutagenesis is a method of protein engineering to create
specific, targeted changes in the protein that may produce a thermo­
stable variant [118]. Many researchers are trying to stabilize the Chase
ABC enzyme using different strategies and approaches. So far, the Chase
ABC structure has been modified through amino acid substitutions to
find out the structure-function relationship and subsequently to
generate thermostable forms of the enzyme (Table 1 and Fig. 2D). In
2013, Nazari at al. showed that relieve of structural strain increases the
thermal stability of the Chase ABC I so that Q140G and Q140A muta­
tions improve both stability and catalytic efficiency of the enzyme
A. Kheirollahi et al. Enzyme and Microbial Technology 172 (2024) 110348

Fig. 5. Challenges in medical application of chondroitinase and applied strategies to overcome these issues.

Table 1
Impact of various mutations on activity and stability of the Chase ABC I.
Chase ABC I mutants Effects Ref

Q140A Improved both activity and thermostability of the enzyme [119]

H700N/L701T Improved thermal stability and decreased catalytic efficiency [120]
R692L/H700A/ Increased catalytic efficiency and decreased thermal stability [120]
Q787A
E138P Improved thermal stability and increased Km value with CS A as substrate [122]
E138D Total loss of the enzyme activity with CS A as substrate [229]
N806Y/Q810Y Improved both activity and thermostability of the enzyme [123]
R660A Total loss of the enzyme activity either with CS A or CS B as substrate [125]
N795A Improved both activity and thermostability of the enzyme; decreased Km value with CS A as substrate and increased Km value with CS B as substrate [125]
W818A Improved both activity and thermostability of the enzyme; decreased Km value either with CS A or CS B as substrate [125]
L679S Improved catalytic efficiency and enzyme half-life [121]
ChABC37-SH3 Improved stability and proteolytic resistance with slight decrease in catalytic efficiency [230]
NΔ5/E694P Improved enzyme half-life and specific activity [231]

[119]. By investigation of a network interaction around Asp689 at the
C-terminal domain of Chase ABC I, it was discovered that the H700N,
L701T, and H700N/L701T mutations increase structural stability
although the catalytic efficiency of the mutants was decreased in com­
parison with the wild type [120]. Besides, it was found that the substi­
tution of Leu679 with Asp and Ser leads to the structural stability of
Chase ABC [121]. In the following, Kheirollahi et al. examined the
rigidifying flexible sites strategy to increase the thermal stability of the
enzyme and showed that E138P mutation increases the half-life of the
enzyme from 8 to 18 min [122]. Results of another study indicated that
the introduction of a cluster of aromatic residues on the surface of the
C-terminal domain increases the activity and stability of the enzyme
[123]. Despite the relative success of the above-mentioned strategies to
increase the thermal stability of the Chase ABC I, adding histidine pairs
and creating His-His interactions could not increase the protein stability
[124]. Chen et al. chose 13 residues for mutagenesis through multiple
sequence alignment of Chase ABC I from P. vulgaris with the homologs
Chase ABC from three organisms (P. hauseri, P. mirabilisbb 2000 and
E. tarda) Chase AC and Chase B from P. heparinus. They found that the
thermostability was enhanced in two mutants, N795A and W818A,
compared to the native protein [125].
In addition to protein engineering, researchers developed other
ways, such as the utilization of stabilizing additives or co-solvents and
deep eutectic solvents, to improve the stability of the Chase ABC. It is
well known that co-solvents can improve the enzyme’s activity and
stability via alterations in the microenvironment of enzymes [126]. In
the case of Chase ABC I, it has been found that the enzyme stability
improves at physiological temperature by adding trehalose to the
enzyme solution [114]. In this regard, Lee et al. demonstrated that the
enzymatic activity of trehalose-stabilized Chase ABC retains for up to 2
weeks post-SCI in vivo, and CSPGs levels remain significantly reduced at
the SCI site for at least 6 weeks post-injury [114]. Based on our exper­
imental data, the Chase ABC I thermal stability increased by glycerol,
sorbitol, and trehalose, as a co-solvent, in vitro [34]. Moreover, it was
found that the co-solvents decrease the rate of both Chase ABC I pro­
teolytic and oxidative inactivation [127]. Among these co-solvents,

7
A. Kheirollahi et al.
trehalose has FDA approval, thus, it can be utilized as a safe stabilizer of
Chase ABC I in vivo. Furthermore, it has been shown that sucrose so­
lution has a considerable effect on Chase ABC stability for about 2 weeks
[128]. Despite the co-solvent mentioned above adding Ca (2 +), aden­
osine, ATP, TNFα, Glu, Gln, Asp, and a combination of them with chase
ABC have no beneficial impact on chase ABC stabilization [129]. In
addition to co-solvents, the effects of natural deep eutectic solvents on
the structure and function of Chase ABCI enzyme was examined and it
was observed that deep eutectic solvents were better media for
improving the thermal stability of Chase ABCI in comparison with the
buffer [130].
5.2. Enzyme delivery
Delivery of the Chase ABC -especially in the case of SCI treatment- is
one of the most important challenges with using it, because this protein
is extremely sensitive and fragile. This enzyme should be used for a long
period and delivered locally; however, a satisfactory system has not
been yet introduced to accomplish this purpose [131,132]. A
well-designed and stable delivery method that could provide the enzyme
at a sustained rate would increase the half-life of the enzyme and sup­
port it against temperature-accelerated degradation, which subse­
quently would decrease the need of frequent usage and consecutive
intrathecal injection [112,133].
5.2.1. Viral vectors
Viral vectors with an insertion of the Chase gene can be injected
directly into the lesion site or transfected into cells that will be trans­
planted into a CNS lesion [113,133–136]. Researchers have modified
the Chase ABC gene of bacterial origin in order to ablate cryptic
N-glycosylation sites and produce active enzymes by mammalian cells
[134,137,138]. Lentiviral (LV) and adeno-associated virus (AAV) vec­
tors have been widely employed to carry the Chase ABC gene into the
CNS [134,137]. Results obtained from the viral delivery of Chase ABC
showed that injection of LV-Chase ABC and AAV-Chase ABC into adult
rat cerebral cortex led to the secretion of active Chase ABC locally as
well as from long-distance axons. Furthermore, it has been observed that
the activity persisted for more than one month [134,137]. Moreover,
direct injection of LV-Chase ABC nearby a spinal cord lesion has been
shown to prevent axonal dieback in corticospinal axons and induce
axonal sprouting in tested animals [134]. In 2015, James et al. demon­
strated that Chase ABC gene therapy –using a lentiviral transfer vector-
could considerably augment the function of the upper limb after cervical
contusion injury [139]. To evade T cell recognition, R. Burnside et al.
designed a novel strategy using a dual lentiviral immune-evasive vector
system for Chase ABC expression based on controlling by a
doxycycline-inducible regulatory switch and this dual vector was
injected into a rat model of contusion SCI [140]. Using this regulable
system, it was reported that following the sustained release of
dox-i-Chase ABC both the horizontal ladder walking task and projection
of dorsal column neurotransmission were improved. However, although
short-term delivery of the enzyme didn’t suffice to improve motor
function, an investigation of the density of vGlut1 + innervation in the
injured spinal cord showed that its long-term delivery improved skilled
hand function due to its ability of long-term CS-GAG digestion [140].
Generally, perineuronal nets (PNNs) are an aggregation form of ECM,
enriched with chondroitin sulfate proteoglycans, hyaluronan, and
tenascin-R, in the brain [141]. Several studies have suggested that the
postnatal maturation of PNNs plays a significant role in neuronal plas­
ticity and neurological disorders [141]. In light of these findings, Cars­
tens KE et al. injected a Cre-dependant AAV vector encoding Chase ABC
into the hippocampus of mice to selectively target tamoxifen-inducible
Cre-expressing CA2 neurons. After a 2-week period of daily treatment
with tamoxifen, PNN loss was observed in the targeted region mean­
while PNNs showed a normal expression in mice receiving the vehicle.
In addition, using this controlled delivery system, mossy fiber sprouting
8
Enzyme and Microbial Technology 172 (2024) 110348
which is an indicator of structural plasticity was increased confirming
the efficiency of this inducible vector [142]. In a further study by Car­
wardine et al. lentiviral vectors encoding active Chase ABC were used to
modify canine olfactory ensheathing cells (OECs) in order to develop a
combination-based therapy that not only has the beneficial effects of a
cell transplant but also allows for sustained delivery of Chase ABC at the
injured siteChase ABC[143]. Since the LV- or AAV-Chase ABC vectors
have a longer-lasting effect and only have to be administered once, they
reduce risks of inflammation, trauma, and infection at the lesion site
[134]. Due to these advantages of the viral delivery, LV- or AAV-Chase
ABC vectors may be considered as a substitute for Chase ABC in
combinational studies. However, it should be noted that the viral de­
livery of Chase might cause new problems since the effects of long-term
or persistent CSPG digestion are unknown [144].
5.2.2. Biomaterials
The use of drug-releasing biomaterials can help to resolve the above-
mentioned challenges in the therapeutic application of Chase. These
polymer-based biomaterials can be either injectable or implantable
[145]. The local and tunable delivery of drug-releasing biomaterials can
prevent adverse repercussions of the systemic delivery of drugs
including a compromised immune system [145]. Biomaterials should be
safe and degrade over time to non-toxic products with minimal immune
response [145]. Hydrogels, particles/tubes, and fibers/conduits are the
most common biomaterial types used in the CNS [146–148]. The
following section describes these biomaterials used in Chase delivery to
the CNS.
5.2.2.1. Hydrogels. Hydrogels, three-dimensional hydrophilic poly­
meric networks, are interesting and trendy bio-compatible composites
for tissue engineering, diagnostics, immobilization, and drug delivery
due to their porosity and compatibility with aqueous environments. The
porosity of these composites can be adjusted by controlling the density
of the cross-linked matrix and this provides a lot of advantages for
loaded molecules such as sustained drug delivery [149]. Having this in
mind, Rossi et al. applied an agarose-carbomer (AC1) hydrogel as a
delivery system for Chase ABC and examined the digestion of decorin, a
scar-like molecule, to investigate entrapped enzyme activity. Using this
delivery system showed enzyme activity for up to 7 days through the
determination of decorin-digested products. Also, this hydrogel didn’t
induce any stable interaction with the enzyme and enzyme denaturation
[150]. Despite these data, designing another delivery system based on
methylcellulose (MC) hydrogel for affinity-based release of Chase ABC
by M. Pakulska et al. [132]. showed better results. Indeed, they modified
the hydrogel by Src homology domain 3 (SH3) binding peptide and also
an SH3 protein to express with Chase ABC protein to investigate whether
or not this strategy can control the enzyme release. The results of this
study showed that this affinity-based MC-hydrogel resulted in sustained
release of active Chase ABC for up to 7 weeks in vitro. Additionally, this
delivery system allows for a controlled release pattern by adjusting the
SH3 peptide to SH3 protein ratio, for example. Similarly, in another
study, a degradable MC hydrogel was synthesized based on an inverse
electron-demand Diels− Alder (IEDDA) reaction, and its potential for
controlled release of Chase ABC was investigated. It was reported that
Chase ABC showed a release pattern for up to 4 days. Additionally, the
cytocompatibility of these hydrogels was confirmed by assessing the
survival and proliferation of neuronal progenitor cells, suggesting a new
strategy for the co-delivery of therapeutic molecules to modulate the
microenvironment of the injured site effectively [151].
Interestingly, H. Hettiaratchi et al. designed a two-pronged approach
for improving Chase ABC stability, as such, they applied site-directed
mutagenesis and PEGylation, which is known to inhibit protein dena­
turation and aggregation, in order to improve enzyme activity and sta­
bility. They reported that N1000G mutation showed better activity and
half-life and additionally, PEGylation of this mutant resulted in further
A. Kheirollahi et al.
improvement of enzyme activity, about 10-fold when compared to un­
modified proteins. Moreover, they also described a methylcellulose
hydrogel as a vehicle for sustained delivery of this modified enzyme in
rat models of stroke and they found a significant decrease in CSPG levels
up to 3 weeks post-injury [152].
Also recently, a study by Raspa et al. [153] used a self-assembling
peptide hydrogel to investigate its potential for immobilization and
sustained release of Chase ABC. Using this delivery system showed a
sustained release pattern of the enzyme for up to 42 days. Additionally,
hydrogels containing the enzyme were injected in chronic SCI lesions
and this resulted in greater BBB (The Basso, Beattie, and Bresnahan)
scores and improved hindlimb locomotor recovery in injured rats.
Overall, despite showing effective function in several studies, there’s a
demand for more studies to confirm hydrogel’s effectiveness in the
repair and regeneration of soft tissues.
5.2.2.2. Collagen. Collagen, the most abundant protein in mammals, is
an attractive polymer due to its low immunogenicity, biocompatibility,
and high degradability. In fact, the hydrophilic nature of this polymer
allows for its high dissolution and release of loaded drugs. Additionally,
as this polymer plays a significant role in ECM, several studies have
investigated its function in the repair of the central and peripheral
nervous systems as well [154]. Therefore, due to its beneficial charac­
teristics, a study conducted by Betül Ahi et al. designed a multilayer
scaffold of collagen/PLGA/Laminin for sustained release of NT-3 and
Chase ABC and investigated its effect on adhesion, proliferation, and
axonal outgrowth in human adipose tissue-derived stem cells (ACSs) and
dorsal root ganglia (DRG) cultures. The results of this study showed that
although the collagen and PLGA layers of this system were able to
improve cell adhesion, proliferation, and outgrowth, in the meantime,
loaded molecules were able to improve their effectiveness [155]. To sum
up, although countless studies about collagen efficacy for tissue regen­
eration are available, its potential for delivering active Chase ABC in SCI
lesions is still a challenge.
5.2.2.3. Silk fibroin films. Silk fibrin (SF), a natural fibrous protein, that
can be extracted from silkworms and spiders is a relatively new strategy
in sustained drug delivery systems for a variety of bioactive molecules
such as drugs, genes, and so on. Its popularity is due to its slow rate of
degradation, structural stiffness, and biocompatibility. In addition,
multiple studies have reported its ability to repair nervous tissues [156].
Meanwhile, a study by N. Sivak et al. investigated the potential of a silk
fibroin-based conduit in the delivery of Chase ABC and glial cell
line-derived neurotrophic factor (GDNF) in a rat model of sciatic nerve
gap injury and animals were followed for 6 weeks. The results of this
study demonstrated that rats receiving conduits releasing both Chase
ABC and GDNF had higher Schwann cell migration to the injured site.
Additionally, axonal regeneration was seen in these rats however there
wasn’t almost a significant difference between this group and those who
were implanted with Chase ABC or GDNF alone [69]. Therefore, more
studies should be conducted to introduce convincing evidence about the
efficacy of these materials in axonal regeneration.
5.2.2.4. Poly (propylene carbonate). Poly (propylene carbonate) (PPC),
a linear aliphatic polymer, can be synthesized from carbon dioxide
(CO2) and propylene. In addition to ease of fabrication, being biode­
gradable and biocompatible, the degradation products of PPC, water,
and CO2, are non-toxic thus making it more attractive for being applied
in a broad range of applications namely drug delivery [157]. Recently, a
study by Xia et al. designed a PPC microfiber for sustained delivery of
dibutyryl cyclic adenosine monophosphate (db-cAMP) and Chase ABC
and investigated not only the in vitro release pattern but also the
implementation of these microfibers in rat models of SCI. The results of
this study showed a stable release profile for db-cAMP and Chase ABC
over a 204-h and 252-h period, respectively. Additionally, applying this
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Enzyme and Microbial Technology 172 (2024) 110348
combination in injured animals led to improved axonal regeneration by
the increase of GAP-43 positive neurons. This result was also associated
with the reduction of glial scar thickness and improved BBB score,
suggesting a higher functional recovery in rats [158].
5.2.2.5. Chitosan. Chitosan, the second largest biopolymer after cellu­
lose, is a natural polysaccharide produced from chitin by a deacetylation
reaction. In recent years, due to some unique properties such as being
biodegradable and biocompatible, chitosan and its derivatives have
attracted a lot of attention for biomedical applications [159]. Addi­
tionally, some studies have shown that owing to having almost similar
composition and structure to GAGs, it shows a minimal immune
response after implantation into the human body, a property that has
proven its non-toxicity [160]. Chitosan has also attracted significant
attention for enzyme immobilization because of having amino and hy­
droxyl groups as its functional groups that improve the interaction be­
tween enzymes and support, a situation that allows for simple
immobilization techniques namely adsorption [161]. A study conducted
by Ni et al. investigated the effect of sustained delivery of Chase ABC
using polypropylene carbonate- chitosan (PPC-CS) micron fibers when
implanted into hemisected spinal cord injury models. The results of this
study revealed that the enzyme showed a ten-day period release profile
from micron fibers in PBS at 37 ◦ C and 5% CO2. Additionally, the
enzyme released from this system resulted in improved functional re­
covery, axonal regeneration, and reduced scar thickness [162]. Another
study by L. Francis et al. assessed the effect of Chitosan-Alginate (C-A)
scaffolds containing chitosan and alginate microspheres for controlled
delivery of Chase ABC and neurotrophin-3 (NT-3). The in vitro release
pattern of these molecules from C-A scaffolds showed an 8-week and a
7-week release profile for NT-3 and Chase ABC, respectively. Also,
molecules released from this delivery system retained their bioactivity,
and culturing DRG neurons in the presence of C-A scaffold containing
NT-3 and Chase ABC improved neurite outgrowth through a growth
inhibitory barrier of CSPGs [163]. However, in vivo, studies need to be
conducted in order to evaluate the ability of these systems for nerve
regeneration.
5.2.2.6. PLGA. Polylactic-co-glycolic acid (PLGA) is an FDA-approved
nanoparticle renowned for biocompatibility, low toxicity, and low-cost
and controllable drug delivery features [164]. Due to these advan­
tages, PLGA is an outstanding polymer-based nanoparticle that has
sparked a lot of attention for its application in various diseases. PLGA
synthesized thorough interactions between glycolic acid and lactic acid
monomers that link together via ester bonds and eventually generate a
negative-charge aliphatic polymer. These nanoparticles integrate into
cells through clathrin-mediated endocytosis or pinocytosis and ulti­
mately enter the cytoplasm. Since PLGA degrades into carbon dioxide
and water via the Krebs cycle it has non-detrimental effects on cells
[165,166]. In this regard, Azizi et al. have loaded Chase ABC I on PLGA
nanoparticles (NPs) and subsequently administered it into SCI-model
rats. According to this study, the BBB score and CSPG degradation
concentration in the immobilized-enzyme-treated group were signifi­
cantly higher compared with the control groups. Additionally, the MTT
assay on OECs confirmed that PLGA NPs are non-toxic [167].
5.2.3. Inorganic nano-materials
Inorganic nano-materials –comprised of noble metals, metal-oxides,
silica-based nanoparticles, and quantum dots have significant physico­
chemical, electromagnetic, and optical features that make them appro­
priate for medical imaging and therapeutic applications. However,
despite their favorable characteristics, their usage is restricted due to
their toxicity and low solubility [168,169].
5.2.3.1. Noble metals. Since noble metallic nanoparticles, like gold
(Au), silver, and platinum. possess remarkable properties, they have
A. Kheirollahi et al. Enzyme and Microbial Technology 172 (2024) 110348

Table 2
Combinatorial strategies focused on chondroitinase.
Combination therapies with Delivery of Chase Models Comments Ref.
chondroitinase

Schwann cells and Osmotic pump In vivo/rat Significant improvements in BBB score. [182]
OECs Increased numbers of both myelinated axons and serotonergic fibers.
OECs OECs transduced In vivo/rat The Chase-generating canine OECs persisted at 4 weeks following [232]
with the Chase ABC-LV vector transplantation.
Increased the number of corticospinal axons caudal.
OECs OECs transduced In vivo/rat A partial return of voluntary movement function. [233]
with the Chase ABC-LV vector Forepaw reaching recovery improvement.
ENSCs LV vector transduction In vivo/rat Injured spinal cords’ cavity area reduction. [234]
The amount of cells projecting axons via the damage site increase.
PPN, BMSCs Direct injection In vivo/rat Considerable axonal regeneration. [235]
Locomotor performance improvement.
oNPCs, methylcellulose biomaterial Direct injection In vitro and in Differentiation of grafted cells to oligodendrocytes increased. [236]
vivo/rat Remyelination of the spared axons increased.
Functional synapse enhancement.
Motor functional recovery improvement.
NESCs, methylcellulose hydrogel Direct injection In vivo/rat Lesion volume reduction. [237]
Chase ABC provides a condition to maintain a greater number of
neurons from transplanted NESCs.
No improvement in functional repair.
hUSCs Direct injection In vivo/rat Functional recovery enhancement. [238]
Increase in NGF and BDNF expression.
ADSCs LV vector transduction In vitro Chase ABC express constantly from ADSCs. [239]
Chase ABC expression rise the chance of ADSCs migration.
AAV-L1 Direct injection In vivo/mice Voluntary movements and functional recovery improvement. [240]
Synaptic plasticity increase.
insulin Direct injection In vivo/rat Neuronal growth and regeneration. [241]
Locomotor and sensor function promotion.
hADSCs Direct injection In vivo/rat Locomotor function improvement. [242]
Lesion area cavity size reduction.
DRG microtransplants, neurotrophin- Direct injection In vivo/rat Axon regeneration [243]
3-expressing lentivirus
PNG Direct injection In vivo/ Adult Axonal regrowth [244]
female cats Synapse formation
OMPCs [245]
MSCs, BDNF Direct injection In vivo/dogs TNF-α, IL-6, COX-2 reduction, BDNF, [246]
β3-tubulin neurofilament augmentation,
BBB score improvement
Lithium Chloride Direct injection In vivo/rat Fortifies Chase ABC function via a Bcl-2-dependent mechanism [247]
NSCs Osmotic pump In vivo/mice Diminished chronic-injury scar and increased iPSC-NSC [206]
differentiation and survival.
pMNs, NT-3, PDGF and NEP1–40 Lipid microtubes embedded in In vivo/rat The combination of pMNs with Chase and NEP1–4- resulted in [248]
fibrin scaffold decreased cell survival and enhanced macrophage infiltration.
PNGs and GDNF Direct injection In vivo/rat Functional axonal regeneration by chronically injured neurons [184]
PNGs and Rheb In vivo/rat Increased the construction of synapses in the spinal cord and [249]
improvement of forelimb function.
Human adipose-derived stem cells Direct injection In vivo/rat Improvement in functional recovery. [205]
NSCs or NT-3 overexpressing NSCs Osmotic pump In vivo/rat Higher neuronal and oligodendroglial [193]
differentiation of NT-3 NSCs.
Enhancement in transplanted cells migration, promoted axonal
remodeling and as well as increased locomotor recovery.
EGF, FGF2, PDGF-AA and treadmill Direct injection In vivo/rat Increased chronic neuroanatomical plasticity with no additional long- [223]
training term improvement in locomotor function.
NGF Alginate In vivo/rat Improved locomotor recovery. [217]
microspheres embedded in
polydioxanone electrospun
filaments
NT-3 Lipid microtubes embedded in In vivo/rat Improvement in locomotor function. [217]
agarose gel
db-cAMP Poly(propylene carbonate) (PPC) In vivo/rat More axonal plasticity, locomotor recovery, and decreased glial scar [114]
electrospun microfibres formation.
KLF7 Viral (LV) transduction In vivo/rat Reduced the net retraction of axons from sites of spinal injury. [158]
Poly(glycerol sebacate) Direct injection In vivo/rat Augmented nerve regeneration and [250]
functional recovery.
Low-level laser (660 nm) Direct injection In vivo/rat Decreased cavity size, increment of myelination and axonal [251]
regeneration.
α-Nogo-A antibody and rehabilitation Direct injection In vivo/rat Increased in sprouting and axon regeneration and enhanced [189]
functional recovery.
Zymosan Direct injection In vitro and in Robust and functional regeneration of sensory axons. [187]
vivo/rat
Oncomodulin and cAMP Direct injection In vitro and in Limited regeneration to injured sensory nerves. [218]
vivo/rat
Clenbuterol Direct injection In vivo/rat Axonal regeneration around the injury site and improved locomotor [220]
function.
(continued on next page)

10
A. Kheirollahi et al.
Table 2 (continued )
Combination therapies with Delivery of Chase Models
chondroitinase
NT3 and NR2D Direct injection In vivo/rat
Liposomal clodronate and Rolipram Direct injection In vivo/rat
been widely used in various research. Among them, gold nanoparticles
have gained a lot of attention. They are, stable, non-toxic, have simple
manufacturing procedures, and exhibit proper optical behavior. Gold
nanoparticles have shown to be a promising choice for delivering
diverse biomolecules and drugs to the target location [170,171].
In this context, Naderi et al. decided to investigate the impact of
different concentrations of Au nanorods on the activity and stability of
Chase ABC I in comparison with the free enzyme. Analysis of circular
dichroism Spectro polarimetry and Fluorescence data showed that the
rigidity of the enzyme raises with a lower concentration of Au nanorods
while it declines at higher concentrations. This gives rise to the theory
that gold nanorods make more chromophores like Tyr and Trp residues
available on the surface and cause the instability of the enzyme. In
addition, this study showed that the presence of gold nanorods can
improve both the substrate affinity and storage stability of the enzyme;
however, it has a negative impact on the enzyme activity that may be a
consequence of gold nanorod inhibition [172].
5.2.3.2. Metal oxide. Metal oxide NPs are multifunctional, stable, less
toxic, and biocompatible drug delivery systems that have widespread
use in medical research. The most common metal oxide nanoparticles
such as Fe3O4, ZnO, CuO, and TiO2 gain a fair amount of attention due
to their magnetic and anti-bacterial effects [173].
In an investigation, Fe3O4 (magnetic) NPs were used as a carrier for
Chase ABC I. The enzyme-linked via ionic, hydrogen, and van der Waals
bonds to the magnetic NPs and their physical connection has been
confirmed with FTIR. The results of the kinetic parameters revealed that
the enzyme affinity to the substrate increased while the specific activity
decreased in the immobilized enzyme.
In another study dextran-coated Fe3O4 was used to evaluate the
Chase ABC I features. The reduced immobilized enzyme’s Vmax and the
increased Km indicate the favorable steric orientation of enzyme active
sites by means of dextran. The nanoparticle also enhanced the stability
of Chase ABC I by 2.5-fold; although, it was inimical to the enzyme
activity. Furthermore, the release pattern of the enzyme reports
demonstrated that 70% of the enzyme was released initially in 9 h
[174].
Additionally, Gao et al. had been seeking a proper approach to
induce the migration of the Schwann cells to the region of the SCI-
induced glial scars. In this scenario, Schwann cells were equipped
with immobilized Chase ABC I gene on polyethyleneimine super­
paramagnetic iron oxide nanoparticles (Chase ABC I/PEISPIONs). The
Chase ABC I degrades CSPGs to make a pathway through the damaged
area. Thereupon, Schwann cells and the resident astrocytes can fuse
together that potentially improve axonal regeneration. Noteworthy that
SPIONs magnetic characteristics were used to control the direction of
Schwann cells toward the desirable region under a magnetic field and
also preserve the Chase ABC I gene against nuclease degradation.
Moreover, they were covered by polyethyleneimine to provide a cationic
surface that is more conducive to the carrier’s cell uptake. Since the
toxicity results point out the safety of the nano-system below 4 µg/ml,
the Chase ABC I/PEISPIONs are regarded as an auspicious candidate for
SCI treatment [175].
5.2.3.3. Silica-based Nanoparticles. Porous silicon (pSi) NPs seem
appealing to scientists owing to their desirable attributes including
biodegradability, biostability, non-toxicity, high loading capacity, and
11
Enzyme and Microbial Technology 172 (2024) 110348
Comments Ref.
Improved axonal sprouting and behavioral recovery. [219]
Reestablished electrical conductivity across the lesion.
Reduced axonal injury and enhanced neuroprotection, plasticity, and [186]
hindlimb functional recovery.
photoluminescence qualities [176]. These features allow them to be an
efficient platform for drug delivery or imaging [177].
In this aspect, green pSi nanoparticles were chosen to entrap Chase
ABC I through electrostatic interaction. The Km value was roughly the
same; nevertheless, the pSi had an adverse effect on the specific activity
and Vmax. These data suggest that the enzyme active sites may engage
during the immobilization processes that also suppress the Chase ABC I
production rate. However, pSi enhanced enzyme thermal stability at
− 20, 4, 25, and 37 ◦ C [176].
In a separate investigation, immobilized Chase ABC I on red pSi ki­
netic parameters yield different results. The study has shown that the
Km rate for the immobilized enzyme was relatively higher than the free
one. Additionally, the activity of the immobilized enzyme on red pSi was
reduced more than the immobilized enzyme on green pSi. The results
also revealed that the red pSi enhanced the Chase ABC I durability at
diverse temperatures. For instance, 70% of the initial activity remained
after 50 min at 37ºC for the immobilized Chase ABC I on red pSi
meanwhile the enzyme activity drops to 60% and 15% for the green pSi
and free respectively. The MTT test found no evidence of cytotoxicity for
red pSi [178].
In consistence with these data, Rezaei et al. carry out research on the
effect of immobilized Chase ABC I on red pSi (Chase ABC@PSi) in an
animal model of MS. Two weeks after the administration of the drug
delivery system they found that CSPGs concertation reduced in the
treatment group in comparison with vehicle and PSi treated groups.
Chase ABC@PSi also increased the number of oligodendrocyte cells in
this study, which helped to promote myelin production. Furthermore,
the reactivity of astrocytes decreased in the treatment group [179].
5.3. Combination-based therapy
Due to the complex nature of SCI pathology, a single-treatment
strategy seems to be insufficient for complete functional recovery. For
this purpose, it may be necessary to apply Chase ABC along with other
effective therapeutic approaches to agitate neuronal regeneration. In
recent years, different methods such as immunomodulation [180],
transplantation of cell and peripheral nerve [181–184], neurotrophic
factors administration [135,185,186], anti-myelin-based inhibitors an­
tibodies (anti-Nogo A) [187], rehabilitation [188], laser therapy and use
of hyperbaric oxygen (HBO) have been combined with delivery of Chase
ABC and revealed more desirable effects [189,190](Table 2).
In addition to increasing axon regeneration, Chase improves migra­
tion of the transplanted cells and their integration into the brain and/or
CSI lesion through degrading the inhibitory action of CSPGs [191–193].
For example, when the axonal growth inhibitory factors were eliminated
by Chase ABC, dopaminergic graft integration in Parkinsonian mice was
remarkably improved [194]. Furthermore, it has also shown that Chase
ABC facilitate the adhesion of chondrocyte and synovial
membrane-derived mesenchymal stem cells to the cartilage disc surface
and partial-thickness chondral defects, respectively [183,195–197]. A
range of cells including neural stem cells [198,199] and glial cells -such
as OECs [200,201], Schwann cells [202,203], and oligodendrocyte
precursor cells [204]- have been investigated for transplantation in the
site of SCI. Recently, the combinatorial effects of Chase and several cell
types were examined in SCI models (Table 2) [182,205–207]. In a study,
the implantation of OECs or Schwann cells into the site of the lesion was
investigated in combination with the prolonged Chase ABC
A. Kheirollahi et al.
administration for four weeks. By employing this combination therapy
graft integration was enhanced and regeneration of axons was improved
across the lesion [208]. In 2015, Lee et al. evaluated the efficacy of the
simultaneous administration of mesenchymal stromal cells and Chase
ABC on chronic SCI and showed that this combination is more effective
in improving clinical signs and neural regeneration [209]. The trans­
planted cells can increase axonal regeneration and promote injured
tissue repair by refilling formed cavities in the site of lesions and
expression of growth factors such as neurotrophins [210,211]. So far,
numerous studies have shown the beneficial effect of various growth
factors such as NT-3, GDNF, IGF, bFGF, TGF-β, BDNF, PDGF, and CNTF
on spinal cord injury [212–216]. The combination of Chase ABC and
neurotrophins omit the inhibitory effect of CSPGs and boost axonal
growth in the new environment with excessive freedom [207]. Hence,
Chase ABC was combined with various growth factors like NT-3 and
NGF and showed significantly improved functional recovery [114,217].
Furthermore, the triple-combination of neural precursor cells, Chase
ABC, and a mixture of PDGF, EGF, and FGF in a clip-compression SCI
model successfully promoted the neural precursor cells’ integration and
differentiation, as well as their migration. Also, the level of GAP43 was
elevated and the plasticity of the corticospinal tract and serotonergic
axons was enhanced, which finally led to improved functional recovery
[191]. In a similar study Hwang et al. combined the poly (ε-capro­
lactone) scaffolds seeded with NT-3 overexpressing neural precursor
cells and Chase ABC in a rat hemisection model and demonstrated that
cell migration, axonal remodeling, and locomotor recovery increase in
the combination group [193].
In some studies, the effect of neuroprotective agents such as zymosan
[218], clenbuterol (a β2-adrenoceptor agonist) [219] and oncomodulin
(Ca2+-binding protein) [220] in combination with Chase ABC is evalu­
ated and their beneficial impacts are proved. In addition, it has been
shown that rehabilitation along with using Chase ABC, in both delayed
and acute stages in the SCI model is more effective [188,221,222]. Be­
sides, specific rehabilitation strategies probably help to the formation of
appropriate connections and adjust the increased plasticity caused by
Chase treatment in the CNS [221]. With an eye toward whether regular
treadmill exercise has a synergistic impact when combined with Chase
ABC and growth factors like EGF, FGF2, and PDGF-AA. Alluin and his
colleagues conducted a study on SCI rat models. This study showed that
the mentioned combinatorial strategy induced considerable structural
plasticity improvement, even so, had no further long-lasting benefits on
the locomotor metrics such as the hindlimb-forelimb connection after
the seven-week recuperation period [223]. In 2013, the impact of
combining rehabilitation, α-Nogo-A antibody, and Chase ABC was
examined in a rat cervical partial SCI model [187]. The N-terminal re­
gion of Nogo-A, called Amino-Nogo-A, is a myelin-associated molecule
and one of the most potent inhibitors of regeneration [224–226]. The
results showed that the combination treatment is more effective; so the
population receiving combination treatment displayed a great recovery
of 80% [187]. One more approach that has been assessed to gain a
beneficial effect of combination therapy was to use GDNF, Chase ABC,
Nogo-A antibody, and PLGA together. GDNF has been utilized to support
neural recovery following SCI since it is a crucial neurotrophic factor.
However, its prompt degradation gave rise to using it with PLGA. It
comes to be more engaging when the research group decides to look into
their synergistic impact with Nogo-A antibody, a corticospinal tract
axonal sprouting accelerative, and Chase ABC. This study showed
PLGA-GDNF, as opposed to GDNF administered directly, clearly
enhanced functional recovery in SCI rats. The combination of
PLGA-GDNF, PLGA-Chase ABC, and PLGA-Nogo worked best to promote
the functional recovery of SCI rats, and the rats in the PLGA-Chase ABC
and PLGA-Nogo groups also had enhanced motor function [227].
Recently, the combination of Chase ABC and laser therapy was
evaluated. The low-level laser (LLL) 660 nm was used with Chase ABC to
investigate their treatment potential together. SCI model rats received
LLL and intraspinal injection of 1 μg Chase ABC treatment for two
12
Enzyme and Microbial Technology 172 (2024) 110348
weeks. Subsequently, the study showed functional recovery, axonal
projections, and myelination improvement in the treatment group. It
also demonstrated the decrease in cavity size in the damaged lesion.
Furthermore, aquaporin 4 (AQP4) expression was reduced which in­
dicates a reduction in edema. Glycogen synthase kinase-3β (GSk3β), a
demyelination factor, was also decreased [189]. It has also been
revealed that the combination of LLL and Chase ABC alleviates allody­
nia, thermal hyperalgesia, and pain-related cytokines such as IL-6 and
BDNF; meanwhile, increased analgesic factors like the glutamic acid
decarboxylase 65-kilodalton isoform (Gad65) and GDNF [228].
The combination of HBO and Chase ABC also appeared to be
contributive in SCI treatment. Following injury significant amount of
free radicals are generated and lead to lipid peroxidation which even­
tually cause neuronal damage. It has also been revealed that the GSK3β
and AQP4 expression increase during SCI which are in charge of CSPG
production and edema respectively. HBO treatment reduces free radical
production and turn the GSK3β and AQP4 expression back to a normal
amount. This study showed that HBO plus Chase ABC treatment would
be effective in neuronal functional recovery [190].
6. Conclusion
According to the observed results in vitro and preclinical studies,
Chase is a promising enzyme in treating various pathological conditions.
For clinical applications, achieving a biochemically stable enzyme is
important because of its thermal instability. In this context, several
studies have shown that the stability of Chase is increased in vitro by
using different approaches; however, it should be investigated in vivo. In
addition, the need for invasive pumps or direct injections can be
removed by delivery of the enzyme through natural, synthetic, and viral
drug delivery systems. Therefore, it seems necessary to focus on the
development of an appropriate delivery system for Chase with the least
side effects. Generally, numerous studies in the fields of enzyme engi­
neering and delivery have been conducted to overcome the Chase
problems in clinical translation. Furthermore, combination therapies
with Chase have also shown promising results, but limited research has
been carried out on Chase drug delivery systems coupled with other
inspiring therapies.
Funding acknowledgments
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
CRediT authorship contribution statement
Asma Kheirollahi: Investigation, Writing – original draft prepara­
tion, Visualization; Solmaz Sadeghi: Investigation, Writing – original
draft preparation, Visualization; Shirin Orandi: Investigation, Writing –
original draft preparation; Kiana Moayedi: Investigation, Writing –
original draft preparation; Khosro Khajeh: Writing – review & editing;
Mehdi Khoobi: Writing – review & editing; Abolfazl Golestani:
Conceptualization, Supervision, Writing – review & editing.
Data Availability
No data was used for the research described in the article.
Acknowledgments
We thank Tehran University of Medical Sciences, Tarbiat Modares
University and the University of Tehran for the support of this
manuscript.
A. Kheirollahi et al.
Conflict of interest
The authors declare that they have no conflict of interest.
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