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J Vet Cardiol. Author manuscript; available in PMC 2023 April 01.
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Published in final edited form as:

J Vet Cardiol. 2022 April ; 40: 2–14. doi:10.1016/j.jvc.2021.05.003.

A review of the underlying genetics and emerging therapies for

canine cardiomyopathies
Lilian Shen, DVMa,*, Amara H. Estrada, DVMb, Kathryn M. Meurs, DVM, PhDc, Meg Sleeper,
VMDb, Christopher Vulpe, MD, PhDd, Christopher J. Martyniuk, PhDd, Christina A. Pacak,
PhDe
aProgram for Applied Research and Development in Genomic Medicine, College of Pharmacy,
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University of Florida, 1225 Center Drive, Gainesville, FL 32610, USA

bDepartmentof Veterinary Clinical Sciences, College of Veterinary Medicine, University of Florida,
PO Box 100136, Gainesville, FL 32610, USA
cDepartment of Clinical Sciences, College of Veterinary Medicine, North Carolina State University,
Raleigh, NC 27607, USA
dDepartment of Physiological Sciences, College of Veterinary Medicine, University of Florida,
1333 Center Dr, Gainesville, FL 32603, USA
eDepartmentof Neurology, College of Medicine, University of Minnesota, 516 Delaware Street
SE, Minneapolis, MN 55455, USA

Abstract
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Cardiomyopathies such as Dilated Cardiomyopathy (DCM) and Arrhythmogenic Right Ventricular

Cardiomyopathy (ARVC) are common in large breed dogs and carry an overall poor prognosis.
Research shows that these diseases have strong breed predilections, and selective breeding has
historically been recommended to reduce the disease prevalence in affected breeds. Treatment of
these diseases is typically palliative and aimed at slowing disease progression and managing
clinical signs of heart failure as they develop. The discovery of specific genetic mutations
underlying cardiomyopathies, such as the striatin mutation in Boxer ARVC and the pyruvate
dehydrogenase kinase 4 (PDK4) and titin mutations in Doberman Pinschers, has strengthened our
ability to screen and selectively breed individuals in an attempt to produce unaffected offspring.
The discovery of these disease-linked mutations has also opened avenues for the development
of gene therapies, including gene transfer and genome editing approaches. This review discusses
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the known genetics of cardiomyopathies in dogs, reviews existing gene therapy strategies and
the status of their development in canines, and discusses ongoing challenges in the clinical
translation of these technologies for treating heart disease. While challenges remain in utilizing

*
Corresponding author: [email protected].
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Conflicts of Interest
The authors have no conflicts of interest to declare in the completion of this manuscript.
 Shen et al. Page 2

these emerging technologies, the exponential growth of the gene therapy field holds great promise
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for future clinical applications.

Keywords
Dilated Cardiomyopathy; Arrhythmogenic Right Ventricular Cardiomyopathy; Gene therapy;
Gene delivery; Genetic testing; Gene editing; Clustered Regularly Interspaced Short Palindromic
Repeats

Introduction
Cardiomyopathies comprise an important group of cardiac disorders in humans and
animals alike. Cardiomyopathies are categorized as Dilated Cardiomyopathy (DCM),
Hypertrophic Cardiomyopathy, Restrictive Cardiomyopathy, or Arrhythmogenic Right
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Ventricular Cardiomyopathy (ARVC) [1]. Hypertrophic cardiomyopathy is the most

common form identified in humans [2] and felines [3], with prevalence approaching up
to 0.4% in humans [1,4,5] and 10–15% in felines [3,6]. In canines, cardiomyopathies show a
strong breed predisposition, with up to 58% of Doberman Pinschers affected by DCM [7,8]
and up to ~25% of Boxers affected by ARVC [9].

Current treatments for cardiomyopathies in dogs aim to prolong the subclinical phase and
treat symptoms of congestive heart failure. Drugs such as pimobendan, a phosphodiesterase
3 inhibitor, and furosemide, a loop diuretic, are two effective treatments currently available
for dogs showing clinical signs, though other drugs such as antiarrhythmics, angiotensin
converting enzyme inhibitors, peripheral vasodilators, and other diuretics are also frequently
utilized. While medications provide improvement in quality of life and extension of the
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occult phase of DCM [10], the efficacy of preclinical treatment of ARVC is less clear
[11,12]. Although the progression of cardiomyopathies can be delayed, no treatment
currently exists which halts or reverses the disease. Prognosis for animals with congestive
heart failure secondary to cardiomyopathy is generally poor, with median survival times as
low as 19 weeks in dogs with DCM [13].

Due to the poor prognosis and limitations of available treatments, increased attention has
been directed toward identifying novel therapies for cardiomyopathy. Multiple genetic
mutations associated with the development of cardiomyopathy have been identified in
humans [14,15] and animals [16,17,18,19,20]. As in humans, genetic disorders in dogs
are amenable to gene therapy approaches which can potentially halt or even reverse the
effects of cardiomyopathy [21,22,23,24,25,26]. This review covers the genetics of clinically
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significant cardiomyopathies in dogs, provides an overview of gene therapy techniques,

and discusses challenges facing the clinical development of gene therapies for canine heart
disease.

Known Genetics of Canine Cardiomyopathies

Increased focus throughout the last two decades on sample populations consisting largely
of single breeds affected by a specific cardiomyopathy has facilitated the identification of

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mutations that appear to be breed-specific. The following section will discuss these genetic
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mutations and modes of inheritance within specific breeds of dogs (summarized in Table 1).

Canine Dilated Cardiomyopathy

Dilated cardiomyopathy is a disease of the cardiac muscle characterized by progressive
systolic dysfunction of the heart resulting in eccentric hypertrophy (dilation) of the
ventricles, predominantly the left ventricle. Individuals affected with this disease may also
present with ventricular or supraventricular arrhythmias. DCM is the second most common
cardiac disease in dogs, behind degenerative valvular disease [8]. Prevalence in the general
canine population is estimated at 0.5% [27]. An increased prevalence is seen within certain
large breeds including the Great Dane, Doberman Pinscher, Portuguese Water Dog, Irish
Wolfhound, Newfoundland, Boxer, Welsh Springer Spaniels, and Cocker Spaniel, among
others. Of these, the Doberman Pinscher is the breed most predisposed to DCM, with
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reported prevalence of up to 58% [7]. Although this breed was the initial focus of inherited
canine DCM studies, recent work has identified genetic alterations in additional breeds
[28,29,30].

Two separate genetic mutations are linked to DCM in Doberman Pinschers. The first
is a 16-base pair deletion located at the donor splice site (5’ end) of an intron in
the phosphodiesterase kinase 4 (PDK4) gene [18]. Inheritance of the PDK4 mutation is
autosomal dominant, and based on a single study, prevalence may be up to 60% with
68% penetrance [18]. Phosphodiesterase kinase 4 is an important regulatory protein in
cardiomyocyte energy metabolism. In the normal, healthy heart, fatty acids are the preferred
energy source, and PDK4 allows for preferential oxidation of fatty acids by inhibiting
glucose oxidation. The identified PDK4 mutation, named DCM1, results in decreased
expression of the PDK4 protein and an energy-deficient state in cardiomyocytes due to
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the lower energy efficiency of glycolysis compared to fatty acid oxidation and a lifetime
of reduced metabolic flexibility in the heart. Phosphodiesterase kinase 4 deficient fibroblast
cells from affected Doberman Pinschers have reduced metabolic compensation capability
during periods of glucose starvation in vitro [31,32].

The second genetic mutation linked to DCM in Doberman Pinschers is a single base pair
change from C to T within the titin gene of affected dogs, resulting in a change in a highly
conserved amino acid from glycine to arginine [19]. The mode of inheritance of the titin
mutation is autosomal dominant, with a penetrance of 47% [19]. Titin is the largest protein
in the body, and it contributes to both passive stiffness and active contraction of the heart
muscle through unfolding and refolding of its numerous domains in response to tension.
The missense mutation identified in Doberman Pinschers, named DCM2, was associated
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with decreased active tension and Z disc streaming [19]. It is hypothesized that changes to
the secondary structure of the protein results in greater ease of unfolding and degradation;
however, the pathophysiology of DCM in relation to the titin mutation is incompletely
understood.

In a recent clinical study DCM2 was identified in more than 50% of the affected dogs, and
20% of affected dogs had both DCM1 and DCM2 [33]. Presence of either the DCM1 or
DCM2 mutation places an individual at a higher risk of developing disease, but it is thought

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that the presence of both mutations heightens risk. No estimation of this combined risk
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exists at the time of this review. Additionally, the two mutations not only involve different
genes but also occur via different mechanisms (i.e. metabolic dysfunction and contractility
defects).

A DCM-linked mutation termed R9H was recently identified in a pedigree of Welsh

Springer Spaniels with a high incidence of left ventricular dilation, poor systolic function,
arrhythmia, and early sudden cardiac death [28]. A single base pair change from G to A
within the phospholamban gene, results in an amino acid change from arginine to histidine
[28]. The mode of inheritance is autosomal dominant, and penetrance is suspected to be
extremely high as all dogs found to carry the mutation developed the disease [28]. An
identical mutation has also been identified in humans, though penetrance is reported to be
much lower [34]. Phospholamban is a key inhibitor of Sarcoplasmic/endoplasmic reticulum
Ca2+ ATPase 2a (SERCA2a), which is responsible for calcium reuptake in cardiomyocytes
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[35]. The R9H mutation results in failure of inhibition of phospholamban and consequently
decreased calcium reuptake by SERCA2a [28].

Evidence of familial autosomal recessive early-onset DCM in Giant Schnauzers has also
been studied, and a 22-bp deletion resulting in a frameshift mutation in RNA-binding motif
protein 20 was identified [36,37]. This mutation has also been identified in humans, though
it is associated with much higher incidence of arrhythmias in humans [38] compared to dogs
[36]. RNA-binding motif protein 20 is involved in the splicing of many important cardiac
genes, and abnormal splicing of titin is considered responsible for the development of
DCM with this mutation [38]. Additionally, aberrant splicing of CAM-kinase and Ryanodine
receptor 2 are thought to occur with this mutation, resulting in a proarrhythmic effect due to
heightened release of calcium from the sarcoplasmic reticulum and L-type calcium channel
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activation [38].

No other genes linked to DCM in dogs have been identified at the time of this review, but
studies have identified potential loci of interest in other predisposed breeds, including the
Portuguese Water Dog [39] and Irish Wolfhound [40].

Canine Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic Right Ventricular Cardiomyopathy is characterized clinically by ventricular
arrhythmias and occasionally systolic dysfunction, and histologically by replacement of
myocardial tissue with fibrofatty infiltrates. While the disease typically affects the right
ventricle, the left ventricle may also be affected, and the high prevalence of ARVC
in Boxer dogs has resulted in use of the term “Boxer Cardiomyopathy”. An 8-base
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pair deletion in the 3’ untranslated region of the striatin gene was identified in Boxer
dogs [41] with autosomal dominant ARVC inheritance, and ~72% penetrance [41]. The
deletion changes the secondary structure of the mRNA which may be linked to the
reduction in striatin expression in affected Boxer dogs. Striatin is a protein localized to
the intercalated discs, which contain gap junctions, responsible for facilitating electrical
conduction between myocardial cells, and desmosomes, responsible for holding myocytes
together during contraction. The role of the intercalated disc in intercellular impulse
conduction and structural integrity may explain the conduction abnormalities and histologic

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changes observed when striatin is disrupted. More severe disease in homozygotes compared
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to heterozygotes has been observed based upon number of ventricular premature complexes
during 24-ambulatory electrocardiogram (Holter monitor) [41].

Other Inherited Canine Arrhythmogenic Diseases

A genetic mutation associated with an inherited ventricular arrhythmia in Rhodesian
Ridgebacks was identified in a family of dogs with history of arrhythmias and sudden death
and no evidence of structural heart disease [30]. A single base pair change from G to A
results in a conserved glycine to serine change in the QIL1 gene [29]. Autosomal recessive
inheritance is proposed, though autosomal dominant with incomplete penetrance cannot be
ruled out [29]. The QIL1 gene product is involved in the Mitochondrial Contact Site and
Cristae Organizing System complex assembly, which is important for cristae stability and
respiratory chain function [42]. The arrhythmias observed with this mutation may be a result
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of irregular respiration and diminished ATP production on action potential production and
myocyte conduction [29].

Inherited ventricular arrhythmias in German Shepherd dogs with no evidence of structural

heart disease may underlie sudden cardiac death [43]. Sudden cardiac death appears to occur
more frequently in dogs younger than 1.5 years and typically occurs during sleep or periods
of rest following exercise [43,44]. Due to these associations, abnormal development of the
autonomic innervation of the heart is hypothesized to trigger early afterdepolarization and
ventricular arrhythmias [44]. Although no causative mutation has been identified to date,
pedigrees suggest either incomplete penetrance of a single gene defect and/or polygenic or
multifactorial inheritance [43].

Genetic Testing in Dogs

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The development of genetic tests has enabled informed breeding and clinical
recommendations in predisposed dogs. North Carolina State University offers a range of
genetic tests for canine cardiomyopathies, including the Doberman Pinscher DCM1f and
DCM2g mutations and Boxer Striatinh mutation. While these tests can guide breeding
and symptomatic screening, it is important to understand their limitations. None of the
cardiomyopathy-linked mutations identified thus far result in 100% penetrance. While the
presence of the mutation increases the risk of developing disease, other factors (exercise,
diet, etc.) influence presentation [45]. Furthermore, genetic variants that remain to be
discovered also contribute to the current manifestation of cardiomyopathies in dogs that
are not attributed to known disease-linked mutations. Despite these limitations, genetic
testing remains an important resource to guide breeding recommendations and estimate the
likelihood of DCM development.
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fNCSU DCM1 Genetic Testing, North Carolina State Veterinary Hospital, Raleigh, NC, USA.
gNCSU DCM2 Genetic Testing, North Carolina State Veterinary Hospital, Raleigh, NC, USA.
hBoxer ARVC Genetic Testing, North Carolina State Veterinary Hospital, Raleigh, NC, USA.

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Current Gene Therapy Techniques

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Gene therapy is a broad term referring to gene based therapeutic approaches for combating
disease, including inherited genetic disorders [21]. Gene therapy for inherited disorders can
be categorized based on approach: 1) correction of a recessive gene deficiency by delivery
of a wild type cDNA (gene transfer), 2) RNA interference of mutant gene transcripts
by delivery of miRNAs (gene silencing), or 3) direct editing of the genome to correct a
mutation or otherwise manipulate the DNA sequence in a way that results in improved gene
function (genome editing). Alternatively, the approach can be used to alter circulating or
cellular protein levels for therapeutic effect (“drug effect”), to treat diseases that are not
inherited or for which the causative mutation is unknown [21]. Each approach requires
careful selection of a delivery vehicle and consideration for an optimal route of delivery to
ensure that the material avoids degradation by the host’s cells, arrives at the desired location,
and achieves adequate transduction levels to produce the desired functional improvements.
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Additionally, consideration must be given to potential off-target effects. Finally, duration of

therapeutic expression (long-term vs. short-term) and potential toxicity or immunogenicity
of either the delivery vehicle or its product are also important treatment considerations. In
this section, we discuss gene therapy approaches for cardiac disease.

Gene Transfer Techniques

The first cardiac gene therapy attempts aimed to promote angiogenesis in coronary and
peripheral artery disease in humans [46]. Intracardiac injections of plasmids encoding
angiogenic growth factors such as VEGF-A and FGF2 were conducted to stimulate
angiogenesis, followed by use of adeno-associated virus (AAV) vectors as delivery systems;
however, after over 150 clinical trials, no successful promotion of angiogenesis has been
achieved [46].
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Similarly, attempts have been made to apply gene therapy in heart failure in humans. One
main area of focus has been the modulation of ionic calcium handling in cardiomyocytes,
which is essential for maintaining normal cardiac function. Sarcoplasmic/endoplasmic
reticulum Ca2+ ATPase 2a (SERCA2a) mediates Ca2+ uptake into the sarcoplasmic
reticulum (SR) in cardiomyocytes, and the expression level and activity of this pump
is reduced in failing hearts [35]. The activity of the SERCA2a pump is inhibited by a
protein called phospholamban, which is deactivated by phosphorylation. One of the first
trials utilizing gene therapy in heart failure involved transfer of 1 × 1013 vector genomes
(vg) of AAV1-SERCA2a into the coronary arteries of failing human hearts. Unfortunately,
despite successes including increase in time to clinical events, decreased frequency of
cardiovascular events, and decrease in mean duration of cardiovascular hospitalizations
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seen in initial trials [47], this treatment failed to improve clinical outcomes, with no
significant differences observed in exercise ability, quality of life, or cardiac biomarker
levels between the treatment and placebo groups during the 12 months of follow-up [48].
Ultimately, analyses of cardiac samples from deceased SERCA2a/AAV1 treated patients
revealed that AAV transduction was lower than that expected for a significant therapeutic
effect suggesting that inefficient transduction was a factor leading to treatment failure.

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A different approach focused on modulation of Ca2+ uptake through overexpression of

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constitutively active I-1c. I-1c is a truncated form of inhibitor 1 (I-1), a protein which
inhibits protein phosphatase 1. Protein phosphatase 1 is a phosphatase which regulates
several cell processes including glycogen metabolism, cell division, muscle contraction, and
signal contraction [49]. Inhibition of this phosphatase by overexpression of its inhibitor I-1c
results in enhanced Phospholamban phosphorylation, which ultimately results in increased
activity of SERCA2a [25]. Intracoronary gene transfer of constitutively active I-1c using
BNP116, a chimeric vector derived from naturally occurring AAV2 and AAV8 capsids, has
shown promise in pigs [23]. High dose (3 × 1012 vg) and low-dose (1 × 1013 vg) injections
of BNP116.I-1c were used, and both groups showed improved cardiac function compared
to control pigs based on echocardiographic assessment two months after experimentally
induced myocardial infarction.

A third approach in attempted to restore Ca2+ homeostasis through overexpression of

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adenylyl cyclase 6, an important cardiac second messenger which increases SERCA2a Ca2+
uptake and reduces phospholamban expression [50]. A single intra-coronary injection of an
adenoviral vector Ad5 expressing Adenylyl cyclase 6 cDNA was administered to human
patients with heart failure, at various doses. Echocardiographic examination was performed
at four and twelve weeks following treatment with adenylyl cyclase 6. Improvement in
ejection fraction was noted in the two highest dose groups at four weeks but not at twelve
weeks, with nonischemic heart failure patients showing the greatest response.

Together, these studies demonstrate the potential and challenges of cardiac gene transfer.
The observed difficulties in transducing target cells have led to the identification and/or
engineering of new AAV vectors for more efficient and specific cell targeting and
transduction.
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Gene Silencing Techniques

Gene silencing involves blocking messenger ribonucleic acid (mRNA) function through
inhibition of protein translation, predominantly via RNA interference and antisense
oligonucleotides (ASO) [51]. RNA interference uses short, synthetic, double-stranded RNA
called small interfering RNA which are specifically designed to pair with a target mRNA
and cause degradation of the mRNA [53,54]. While antisense techniques work similarly,
ASOs are typically single-stranded RNA molecules which complement the target mRNA
and cause either degradation of the target mRNA or blocking of translation through different
mechanisms such as modification of splicing and steric hindrance [54,55].

A recent example of the application of ASO technology in canine disease is canine

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Duchenne Muscular Dystrophy (DMD), a disease characterized by progressive degeneration

of skeletal and smooth muscles, including the heart. Dystrophin is a large scaffolding protein
which links the cytoskeleton with the sarcolemma of muscle tissue and helps to preserve
myofibril integrity [54]. Loss of this structural function results in the muscle weakness
and atrophy observed in individuals affected with DMD. Thousands of mutations within
the dystrophin gene have been linked to DMD, and many of these are concentrated in the
region of exons 45–50, and cause a frameshift that results in failure to express a functional
dystrophin protein due to premature stop codons [26]. An ASO was designed to bind near

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the splice acceptor site of exon 51 and mask splice signals, resulting in skipping of exon
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51 and restoration of the open reading frame with improved production of a truncated but
functional dystrophin protein in canine patients [55].

Additionally, ASO technology has been used in an in vitro study of human titin-based DCM
[56]. A 2-bp insertion in exon 326 of the titin gene produces a premature stop codon in
one form of inherited DCM in humans, resulting in a truncated protein. Through lentiviral-
mediated ASO transfection of human iPSC-derived cardiomyocytes, exon 326 skipping
was achieved, with associated improvement in protein expression, sarcomere structure, and
contractile performance [56].

Gene Editing Techniques

While the studies discussed above show the promise of gene transfer for heart failure,
the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) gene
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editing techniques has broadened the scope of gene therapy beyond gene delivery to
include the transfer of factors able to correct genomic variants. Gene editing technologies
take advantage of components of natural bacterial adaptive immune response mechanisms
in order to activate, deactivate, or alter specific genes [24,59,60]. Clustered regularly
interspaced short palindromic repeats and CRISPR associated protein 9 (Cas9) based
approaches use short RNA sequences called guide RNAs (gRNA) in combination with the
Cas9 endonuclease to identify and cut specific nucleic acid sequences [Figure 1]. A short
sequence of DNA called a protospacer adjacent motif located near the target sequence is
also required for Cas9 cleavage [59]. Numerous variants of Cas9 enzymes are found within
different bacterial species [60], and subsequent directed evolution of Cas9 enzymes has led
to the capability to target almost any DNA sequence [60,62,63].
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Once a targeted double-stranded cut has been made, the DNA must be repaired, and this
occurs via one of two mechanisms: non-homologous end joining or homology directed
repair (HDR) [61]. Non-homologous end joining is a fast, efficient method of DNA repair
in which the ends of a double-stranded break are ligated [Figure 2a]. This mechanism may
occur in any phase of the cell cycle but can be error-prone and generates genetic insertion/
deletions [62]. In contrast, HDR relies on a DNA template in order to enable precise repair
of a break and preserve genetic integrity [63] [Figure 2b]. This process, however, is thought
to be mostly limited to the S- or G2-phase of the cell cycle, thus limiting efficiency of HDR
in mature non-replicating cells [64].

A recent example of the application of gene editing technology in canine disease is that
of DMD, mentioned earlier. Building on the discovery that ASO-mediated exon skipping
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can successfully restore the open reading frame, a CRISPR/Cas9 construct was designed to
edit the splice acceptor site of exon 51, resulting in exon 51 skipping and production of
functional dystrophin in dogs [26]. Using an AAV9 vector, 1.2 × 1013 vg was injected into
one of the cranialis tibialis muscles of study dogs, resulting in restoration of approximately
60% of wildtype levels of dystrophin in the treated muscle and only 2% of wildtype levels
of dystrophin in the untreated muscle 6 weeks after treatment. Systemic high-dose injection
of 1 × 1014 vg using an AAV9 vector was demonstrated to restore up to 70% of wildtype

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levels of dystrophin expression in skeletal muscle and up to 92% in cardiac muscle 8 weeks
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following treatment.

These previous gene therapy studies have guided ongoing research and will help to
further inform future studies. Failures in effective transduction of target cells have led
to creative approaches to generate engineered serotypes with the capacity to target and
transduce specific tissues types more efficiently [65]. The successful restoration of the open
reading frame in DMD using either an antisense oligonucleotide or CRISPR gene editing
demonstrates the repertoire of effective strategies available to present day scientists. In
combination with improved technology and an increased understanding of inherited disease,
the review and critical assessment of previous experiments allows us to continue to build this
new branch of science and may lead to profound improvement in the care and quality of life
of patients of all species.
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In Vivo Gene Delivery Techniques

While many different inexpensive and efficient methods of gene delivery to cells in vitro
and ex vitro exist, delivery to cells and tissues in vivo is more challenging. In vivo
delivery is clinically necessary, but successful transduction can be difficult to achieve due to
interactions with non-target cells and the host immune system [66]. The ideal delivery vector
will be non-immunogenic with high specificity for the target cell or tissue types, and result
in sufficient transduction and transgene expression levels to produce the desired therapeutic
effect. Gene delivery systems are typically categorized as viral vs. nonviral. While nonviral
delivery systems are typically less immunogenic, their lower ability to effectively target
mature cells such as cardiomyocytes and reduced persistence over time make these systems,
with the possible exception of nanoparticle delivery systems [69,70,71], less effective for
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cardiac gene therapy. Thus, this review will focus on commonly used viral vector systems.

Typical viruses used as vectors for gene delivery include adenovirus, adeno-associated virus,
lentivirus, human immunodeficiency virus, and herpes simplex virus [70], among others.
Portions of plasmids containing DNA/RNA and/or CRISPR/Cas9 constructs (gene editing
materials) are packaged into the virus, which delivers these materials to the target cell for
expression. Several different virus strains have been identified with different characteristics
including varied expression duration (long-term vs. short-term), diverse somatic cell line
targets, and different packaging capacities.

Adenovirus
Adenovirus, a medium-sized non-enveloped virus, is a widely used viral vector in
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gene transfer. Adenoviruses naturally cause diseases in mammals; therefore, it was not
unexpected that the original adenovirus vectors evoked a strong immune response from
hosts. Second- and third-generation adenoviruses containing deletions were engineered to be
less immunogenic [70]. While genetic material can gain entry into the nucleus of infected
cells and be transcribed using such a system, integration of the donor DNA into the host
genome does not occur and therefore short-term expression is achieved.

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Adeno-associated virus
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Adeno-associated virus is a small non-enveloped virus in the Parvoviridae family. It is

capable of infecting both dividing and non-dividing cells and has been found to have low
pathogenicity and low toxicity, although repeated administration of AAV vectors triggers
an immune response [66]. Adeno-associated virus has evolved to enter cells through
interactions with surface molecules on target cells. Differences in sugar-binding preferences
and secondary receptors confer the varying tissue selectivity that distinguishes the different
viral serotypes (AAV 1–9), and as a result one variant may be chosen over another in
order to preferentially transduce a particular cell type [71]. The recombinant AAVs (rAAVs)
used in gene delivery studies retain only the inverted terminal repeats of the original viral
genome. While rAAVs do not undergo host genome integration, they do persist for long
periods of time as episomes in the nucleus of cells [72]. Due to the lack of host genome
integration, repeated administration may be necessary in some gene therapy approaches
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where the target cell population is replaced over time, as the rAAV episomes become diluted
out among daughter nuclei with cell division.

One of the greatest limitations of this viral vector is the small packaging capacity of
approximately 4.8 kilobases [24,75]. This is a challenge for delivery of large genes (e.g.
dystrophin or titin) and also for CRISPR gene editing approaches which require packaging
of a Cas9 gene and gRNA in addition to template DNA for HDR. As an example, wildtype
Cas9 derived from Streptococcus pyogenes (spCas9) is approximately 4.1 kb, leaving little
room for addition of gRNA and template DNA [24]. One method of circumventing this issue
is the use of dual vectors, with one AAV containing the Cas9 enzyme and another containing
the gRNA and DNA template [74]. This approach, however, requires that each target cell be
transduced with both AAV vectors for the desired gene editing to occur. Another method of
adjusting to the size constraints of AAVs is using smaller Cas9 variants. While spCas9 is the
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original Cas9 enzyme found in Streptococcus pyogenes, other Cas9 enzymes with differing
properties including smaller sizes have been isolated from other bacterial species, ranging
from Francisella novicida (~1.6 kb) to Campylobacter jejuni (~984 bp) [61]. Yet another
approach is split-Cas9 systems utilizing split-inteins, which can be thought of as protein
introns. Cas9 halves connected to split-inteins may be administered separately, and trans-
splicing of inteins allows for reconstitution of the complete Cas9 following transfection [75].

Retrovirus
Retroviruses are enveloped viruses containing an RNA genome. They are particularly
useful for long-term expression due to integration of DNA into the host genome [76].
As random genome integration of retroviruses is problematic, safer vectors designed for
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targeted integration are being developed to minimize undesired host mutagenesis. There
is also concern regarding the potential for pathogenicity when using this viral vector, as
replication-competent cells may be produced. Another significant drawback of this type of
viral vector is the inability to infect nondividing cells; however, lentivirus, a specific type of
retrovirus, is capable of infecting both dividing and nondividing cells.

Lentivirus is a type of retrovirus mainly derived from human immunodeficiency virus

1 which differs from other retroviruses in its ability to translocate across the nuclear

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membrane and therefore transduce nonreplicating cells [77]. This ability makes lentiviruses
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particularly useful in gene editing of mature somatic cells such as cardiomyocytes. Safer
lentiviral vectors have been engineered through removal of accessory virulence factors and
splitting of the viral genome into separate plasmids, which reduce the risk of creating
recombinant viruses [78]. Insertion of lentiviral vectors near oncogenes in the host genome
and subsequent tumor formation remains a concern.

Although not covered in this review, multiple additional viruses have been adapted as
vectors in gene therapy systems [72,76]. The continued development of vectors with
improved ability to target and transduce specific cells will enable future genetic therapeutic
approaches.

Challenges in Gene Therapy

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While gene therapy holds great promise for the treatment of inherited and non-inherited
disorders, there are several challenges in the potential clinical application. Gene transfer and
gene editing both require vectors to carry enzymes and/or genetic material to target cells. As
discussed in the previous section, size constraints of certain vectors are an issue particularly
when attempting to transfer large genes (gene transfer) or when using larger Cas9 enzymes
(gene editing) [48,72,24,76]. In the case of gene transfer, the use of minigenes (truncated
but functional forms of a gene) shows promise [79]. In gene editing, packaging of the Cas9
enzyme and gRNA/DNA template into separate plasmids, use of smaller Cas9 variants, and
use of split-Cas9 systems are potential options [63,24,76].

As discussed previously, some vectors that insert into the host genome also carry the risk
of oncogenesis [80,82,83,84]. Avoidance of these types of vectors is not always possible,
especially if viral integration into the host genome for long-term expression of a gene is
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desired. The alteration of existing viral vectors including deletion of accessory virulence
factors in order to reduce oncogenesis and risk of recombination is another potential
solution, though the risk is not eliminated [80].

Immunogenicity is a large challenge in the clinical application of gene therapy since

many commonly used viral vectors naturally infect humans and animals and therefore
have the potential to stimulate both innate and adaptive immune responses [85,86]. An
immune response against a gene therapy vector may eliminate the vector and/or transfected
cells, potentially reducing the duration and intensity of transgene protein expression [85].
Treatments must be optimized to use the lowest possible therapeutic dose to achieve
adequate transduction of target cells and avoid stimulation of the host immune response,
as the latter can be fatal [86,87].
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Clustered regularly interspaced short palindromic repeats technology continues to improve

exponentially, with the number of known Cas9 systems and the repertoire of delivery vectors
quickly expanding. Progress thus far gives hope for the development of new, more effective
treatments for inherited diseases. However, it is important to note the unique limitations and
challenges faced by this new branch of therapeutics. First, efficiency of gene editing systems
is a limiting factor. While the existence of many Cas9 variants theoretically enables targeting

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 Shen et al. Page 12

of almost any portion of the genome, the insertion of specific DNA sequences is limited
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by HDR, which is much lower in efficiency compared to the more error-prone process of
non-homologous end joining. As discussed in the previous section, HDR is significantly
limited in nonreplicating cells such as cardiomyocytes, reducing the efficiency of gene
editing. Additionally, Cas9 enzymes have variable specificities, depending on the variant
and the sequence to be targeted. Off-target endonuclease activity may result in the creation
of undesired mutations and unpredictable consequences. Due to this risk, extensive in vitro
testing and optimization is essential.

Many inherited diseases including inherited cardiomyopathies are multifactorial; that is,
they are influenced by lifestyle and environment in addition to typically involving several
different genes, some of which remain unidentified [16,17,18,19,20,40,41,48]. Gene editing
of specific targeted mutations will likely not provide a single treatment which can be broadly
applied to all affected patients with diseases which are known to be caused my many
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unique mutations (e.g. human hypertrophic cardiomyopathy). However, the smaller number
of uniform mutations identified in dogs may make this species more amenable to genome
editing, and gene delivery to address molecular abnormalities (such as calcium cycling)
remains an alternative approach.

The incomplete penetrance of many mutations linked to inherited diseases also makes it
difficult to predict which individuals will develop the disease phenotype and to what extent.
Here the question arises as to which patients should be treated with gene therapy and when
such a treatment should be performed. It is reasonable to assume in the case of inherited
cardiac disorders, treatment should be encouraged prior to the onset of irreversible structural
and hemodynamic changes; however, the variable length of the occult stage of some diseases
makes this determination difficult.
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Finally, the determination of whether a treatment was a success (i.e., the target gene was
successfully edited as intended) or a failure can be difficult. This uncertainty is linked to
the multifactorial nature of disease and variation in disease phenotypes among affected
individuals. Successful genetic treatment of a patient may still result in clinical disease due
to a separate, unidentified mutation or other environmental factors. Conversely, a treated
patient may fail to develop an inherited disorder, but determination of treatment success
cannot be assumed since it is difficult to predict if this patient would have ever developed
clinical disease even without genetic therapy. Prospective studies evaluating treatments must
be sufficiently powered to overcome these obstacles in determining success or failure, and
natural history studies are essential to the design of clinical trials with appropriate outcome
measures for the target patient population.
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Repeated genetic screening using non-target cells (e.g., cheek swab) from a treated
individual would be expected to continue showing a mutant genotype due to the theoretic
cell type specificity of gene editing (i.e., only cardiomyocytes should show genetic changes
after treatment). Genetic screening of the target cell type following treatment would help
to confirm treatment success; however, in the case of cardiomyopathies, repeated screening
is not practical antemortem, as cardiomyocytes are not routinely sampled in live animals.
Another method of gauging treatment success may include selecting patients which have

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 Shen et al. Page 13

developed some signs of structural disease (e.g., left ventricular (LV) thickening and
Author Manuscript

diastolic dysfunction in hypertrophic cardiomyopathy or left ventricular dilation and systolic

dysfunction in DCM), then performing follow-up examinations following treatment to
monitor disease improvement or progression.

Conclusion
The discovery of specific genetic mutations linked to certain cardiomyopathies has
strengthened our ability to screen and estimate risk of cardiac disease development in
certain breeds. While the availability of commercial tests for these genetic mutations is
helpful, incomplete penetrance and the multifactorial nature of cardiomyopathies continues
to complicate interpretation of test results. Genetic testing should be viewed as a method to
assess risk of disease in a breeding animal or beloved pet rather than a method of diagnosis,
and interpretation of results under the guidance of a qualified professional is recommended.
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The discovery of these disease-linked mutations has also opened avenues for seeking
different forms of treatment. While gene therapy holds promise, challenges such as
identification of vectors with adequate packaging capacity and efficient target cell
transduction must be overcome to make clinical application widely feasible. Additionally,
the complexity of breed-specific mutations necessitates the investigation of different
approaches as opposed to a universal treatment to optimize treatment success. While
significant limitations and challenges in genetic editing have been identified, the continued
exponential growth of this branch of research shows great promise for future clinical
applications.

Funding
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The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of
this article: This work was supported by the National Institutes of Health [grant number T32 HG008958].

Abbreviations
AAV adeno-associated virus

ARVC arrhythmogenic right ventricular cardiomyopathy

ASO antisense oligonucleotide

Cas9 CRISPR associated protein 9

CRISPR clustered regularly interspaced short palindromic repeats

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DCM dilated cardiomyopathy

DMD Duchenne muscular dystrophy

gRNA guide ribonucleic acid

HDR homology directed repair

mRNA messenger ribonucleic acid

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 Shen et al. Page 14

PDK4 phosphodiesterase kinase 4

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SERCA2a sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a

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Figure 1:
Schematic diagram of the Clustered regularly interspaced short palindromic repeats
(CRISPR) and CRISPR associated protein 9 (Cas9) system. Cas9 (orange) forms a complex
with a guide RNA (gRNA, blue), creating a sequence-specific endonuclease. The gRNA
recognizes a target sequence (light green) ending with a 3’ protospacer adjacent motif
sequence, enabling double-stranded cleavage of the target DNA.
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 Shen et al. Page 20
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Figure 2:
Schematic diagram of possible double-stranded break repair mechanisms. (a) Non-
homologous end joining is a fast, efficient pathway which frequently results in insertions
and/or deletions. (b) Homology directed repair is a slower pathway which uses a
deoxyribonucleic acid (DNA) template, allowing for precise repair and possibly DNA
modification.
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Table 1:

Summary of known breed-specific genetic mutations linked to cardiomyopathies in canines.

Mode of
Shen et al.

Name Breed affected Disease Gene involved Mutation type Biological result inheritance Penetrance
Doberman Phosphodiesterase kinase Altered cardiomyocyte metabolism with Autosomal
DCM1 pinscher Dilated Cardiomyopathy 4 (PDK 4) 16 base pair deletion preferential glucose oxidation dominant 68%

Incompletely understood; hypothesized

Doberman Single base pair (missense) changes to secondary structure resulting Autosomal
DCM2 pinscher Dilated Cardiomyopathy Titin change from C to T in titin unfolding and degeneration dominant 47%

Arrhythmogenic
Right Ventricular Altered electrical conduction and Autosomal
Striatin Boxer Cardiomyopathy Striatin 8 base pair deletion structural integrity between myocytes dominant 72%

J Vet Cardiol. Author manuscript; available in PMC 2023 April 01.

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