Dooley et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03156-x(2024) 21:183

Journal of Neuroinflammation

Open Access

REVIEW

The potential of gene delivery for thetreatment of traumatic brain injury
James Dooley1*, Jasmine G. Hughes1,2, Edward J. Needham2, Katerina A. Palios1 and Adrian Liston1
Abstract
Therapeutics for traumatic brains injuries constitute a global unmet medical need. Despite the advances inneurocritical care, which have dramatically improved the survival rate for the ~ 70 million patients annually, few
treatments have been developed to counter the long-term neuroinflammatory processes and accompanying
cognitive impairments, frequent among patients. This review looks at gene delivery as a potential therapeutic
development avenue for traumatic brain injury. We discuss the capacity of gene delivery to function in traumaticbrain injury, by producing beneficial biologics within the brain. Gene delivery modalities, promising vectors andkey delivery routes are discussed, along with the pathways that biological cargos could target to improve longterm outcomes for patients. Coupling blood-brain barrier crossing with sustained local production, gene delivery
has the potential to convert proteins with useful biological properties, but poor pharmacodynamics, into effective
therapeutics. Finally, we review the limitations and health economics of traumatic brain injury, and whether future
gene delivery approaches will be viable for patients and health care systems.
Keywords Gene delivery, AAV, Neuroimmunology, TBI

Main text
Traumatic brain injury (TBI) is a major cause of global
morbidity and mortality. Causes of TBI range frommotor vehicle accidents, falls, and community violence,to sports and workplace-related injuries. The annual
global incidence of TBI is approximately 70 million [1],with lifetime risk estimates as high as 50% [2]. Of patients
admitted to intensive care with TBI, over 20% die within
the first six months of injury, and a further 20% rely onothers for help with basic activities of daily living; only aminority ever achieve a full recovery [3]. Perhaps more
surprising is that nearly one-third of patients with mild

*Correspondence:
James Dooley
jd2045@cam.ac.uk
1
Department of Pathology, University of Cambridge, Cambridge, UK
2
Department of Clinical Neuroscience, University of Cambridge,
Cambridge, UK

TBI (who did not require hospital admission) have notmade a full recovery by six months post-injury [3].
TBI comprises of two distinct components, the primary
and the secondary injury. The primary injury occurs
after mechanical forces act on the brain, generating diffuse axonal injury and/or contusions (bruising). Acutely
following injury, neuronal ionic concentrations are dramatically perturbed, generating excess glutamate release,excitotoxicity, disruption of calcium homeostasis andmitochondrial dysfunction leading to oxidative damage,and cell death [4, 5]. Additionally, the blood-brain barrier(BBB) is disrupted, leading to periods of ischemia, which
can create a severe energy crisis. Of note, cerebrovascular dysfunction appears to persist to chronic timepoints[6, 7]. The secondary injury follows and exacerbates tissue damage by a variety of neurotoxic biochemical events
starting within minutes and persisting for years after the
initial insult. In parallel to these events, neuroinflammation is initiated and maintained into a chronic stage.

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Dooley et al. Journal of Neuroinflammation(2024) 21:183

Microglia are the first responders to injury, followed by
infiltration of peripheral immune cells: first neutrophils,
then macrophages alongside components of the adaptive
immune system [8]. While the initial immune responseis critical for clearance of neuronal debris, failure of theimmune response to resolve over time can have deleterious consequences.
In the years following a moderate or severe TBI, or
sometimes after a series of mild TBIs [9, 10], the brain
may undergo pathophysiologic changes that are challenging, and often impossible, to conclusively detect in living
subjects using presently-available diagnostic techniques.
These changes may include alterations in neuronal structure and function, abnormal protein accumulation, andneuroinflammation. Despite advancements in neuroimaging and biomarker analysis, subtle and diffuse brain
damage resulting from TBI can remain elusive, underscoring the need for further research into more sensitive
diagnostic modalities [11]. In the long term, a majority ofpatients with TBI continue to show a persistent impairment in the ability to complete activities of daily living 5
years after injury, with fatigue, difficulties in cognition,
memory or communication problems, or issues withdepression or aggression [12]. Furthermore, it is now
established that TBI, in particular severe TBI, plays a significant role in the aetiology of several neurodegenerative
diseases, substantially increasing the risk of developing conditions such as Alzheimer’s disease [11], Parkinson’s disease [13], amyotrophic lateral sclerosis [14], andchronic traumatic encephalopathy [15] later in life.
Neuroinflammation in TBI and the potential for new
therapeutics

A key driver of these long-term pathological impacts isthe secondary neuroinflammation, which can extend
for many years post-injury. These inflammatory processes involve immune cell activation, cytokine release,and blood-brain barrier disruption propagating a positive feedback of immune response [16]. The chronicityof these effects is demonstrated by chronic activation of
microglia in rodents [17–19], non-human primates [20],and humans [21]. Importantly, this chronic inflammation is closely associated with neuronal and white matterdegeneration in both humans and mice, which has been
demonstrated to spread to uninjured regions in mice [17,18, 21]. Accompanying the activation of microglia is therecruitment of circulating inflammatory cells and macrophages into injured brain tissue. This dual mechanism
underscores the role of chronic inflammation in TBI
pathophysiology across species [17, 22–24]. The durationof the neuroinflammatory response has a detrimental
impact on the long-term outcomes of TBI, with inflammatory biomarkers correlating with pathological outcomes. These biomarkers exhibit a temporal continuum,

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with a shifting contribution by diverse pathways, reflecting the evolving pathophysiology of TBI [25].
The cascade of inflammation over the acute-subacuteperiod following TBI is highly dynamic. Neutrophils are
the first peripheral immune cell to infiltrate damaged tissue to promote clearance of cellular debris, but concomitantly increase BBB disruption and vasogenic oedema
via increased production of matrix metalloproteinases
in humans and rodents [26, 27]. Moreover, neutrophils
undergo NETosis, releasing extracellular traps to facilitate debris clearance which can cause collateral damage of neighbouring neurons [28]. In the following days,
monocytes and components of the adaptive immune system are recruited to the injury site where they both help
and hinder the reparative process. Rag1 knockout micedemonstrate reduced lesion sizes at subacute timepoints
following TBI suggesting T and B lymphocyte involvement in TBI may be detrimental [29]. Others have identified heightened inflammation associated with worse
neurological outcomes in mice deficient in B cells following TBI, while the opposite was true for depletion of
pro-inflammatory, cytotoxic CD8 T cell subtypes [30].
Enhancement of endogenous regulatory T cells, which
are anti-inflammatory in nature, reduced lesion sizes tocomparable levels to Rag1 knockout mice [29]. These
data suggest specific immune cell subsets can contributeto injury pathology to differing degrees during the subacute post-injury milieu.
Microglia and astrocytes appear to be the predominant drivers of inflammation from subacute to chronic
timepoints post-TBI, thereby providing a large therapeutic window for possible intervention. Both humanand murine microglia undergo considerable changes intheir morphology in response to TBI, with most becoming bushy-like or amoeboid in appearance, reflecting
an activation state comparable to macrophages [17,21]. Microglia and macrophages are considered to play
similar roles in the immune response to TBI, including
phagocytosis of myelin and neuronal debris to facilitate
neuro-reparative processes. Additionally, preclinicalmodels of TBI have identified alternative activation statesof microglia and macrophages with an increased prevalence of pro-inflammatory “M1-like” vs. pro-reparative“M2-like” phenotypes in chronic TBI [31–34]. In excess,M1-like phenotypes are detrimental to recovery and areassociated with the production of reactive oxygen species(ROS) and inflammatory cytokines, culminating in theactivation of cell death pathways [17]. Similarly, whilst
not classically considered immune cells, astrocytes may
also be classified into purportedly neurotoxic A1-like vs.
neuroprotective A2-like phenotypes [35]. Both A1-likeastrocytes and M1-like microglia are robustly associatedwith complement expression and appear upregulated inrodent TBI models [36, 37]. However, glia often display

Dooley et al. Journal of Neuroinflammation(2024) 21:183

concurrent expression of M1/A1- and M2/A2-like markers in preclinical TBI, suggesting a spectrum of activation
states is most likely [31–34, 38, 39]. The importance of
mixed glial phenotypes following brain injury may partially explain why global deletion of microglia or astrocytes can exacerbate neurological sequalae in rodent
models of neurotrauma [40, 41]. Conversely, transient
depletion and subsequent repopulation of microglia
appears to be beneficial following murine TBI [19, 42, 43].
Here, repopulating microglia adopt a neuroprotective
phenotype associated with reductions in NLPR3 inflammasome activation and interferon signalling, alongsideenhancing IL-6-mediated neurogenesis and repair following TBI. This highlights the complexity and importance of specific glial subsets in the chronic secondary
injury following TBI, rather than total cell numbers.
Ultimately, the immune response to TBI is spatiotemporally complex, with elements crucial for repair andprevention of further damage. However, the immune
response can quickly become uncontrolled and dysregulated, leading to chronic activation of immune cells andexacerbation of the initial injury. Identifying the immune
cell subsets that can be detrimental in excess, what timepoints they exhibit neuroprotective versus neurotoxic
qualities, and what inflammatory cellular crosstalk takes
place will be important for the development of inflammation targeted therapeutics. Optimal therapeutics for TBI
would attenuate neurotoxic immune subsets whilst maintaining and enhancing endogenous reparative immune
phenotypes.
Despite the substantial unmet medical need posed
by TBI, treatment options are currently limited. Developing effective therapies to address the complex anddiverse consequences of TBI remains a significant challenge. Neurocritical care focuses on maintaining physiological stability and averting secondary injury. Surgicalinterventions like decompressive craniotomy and ventriculostomy are valuable for mitigating the impact ofbrain swelling by reducing intracranial pressure [44, 45].
Following the emergency care period, medical intervention relies on symptom management and rehabilitation.
The advances in neurocritical care over the past decades
have seen dramatic improvements in survival following
moderate and severe TBI [3]. However, while effective in
addressing immediate concerns, these procedures often
do not adequately target the ongoing secondary effects
of TBI. Thus, despite their efficacy in managing certain
aspects of TBI, additional interventions or therapies
are needed to address the broader spectrum of cognitive deficits and neurological sequelae associated with
the TBI. The rise of biologics as effective modulators ofcomplex physiological processes, in particular immune
regulation, holds out the hope for drugs capable of mitigating the pathological processes of TBI. The kinetics andPage 3 of 15

biodistribution of biologics are, however, poorly suitedfor the chronic processes occurring post-TBI. Gene delivery systems are reviewed here as a potential strategy tocouple the potency of biologics with the sustained local
production enabled by gene delivery vectors.
Neuro-delivery of therapeutic cargos

The challenge of generating effective TBI therapies partly
stems from the difficulty in delivering therapeutics to thebrain to target neuroinflammation and promote neurorepair. The BBB presents a major hurdle, impeding
the passage of therapeutics due to size and hydrophilicity constraints, complicated by BBB dysfunction andglial scar formation post-TBI [46]. The inflammatory
microenvironment further complicates drug delivery byaltering vascular permeability and promoting glial scarformation. Another issue to be overcome for biologics inparticular, is the incompatibility of the prolonged kinetics
of post-TBI pathology compared to the half-life and costof biologics. Innovative delivery systems are, however,being developed, which enable the production or deliveryof therapeutic proteins to the brain. These approaches
include:• 	 Targeted drug delivery: Utilizing ligands or
antibodies that specifically bind to receptors ortransporters expressed on brain endothelial cells tofacilitate targeted delivery of therapeutics across the
BBB.
• 	 Intranasal delivery: Exploiting the olfactory andtrigeminal nerve pathways to bypass the BBB
and deliver therapeutics directly to the brain via
intranasal administration.
• 	 Focused ultrasound: Using focused ultrasound in
conjunction with microbubbles to transiently disrupt
the BBB at targeted brain regions, enabling enhanced
delivery of therapeutics.
• 	 Chemical modifications: Engineering small moleculedrugs with chemical modifications to enhancetheir ability to cross the BBB, such as increasing
lipophilicity or altering molecular size.
• 	 Bioengineered carriers: Developing bioengineered
carrier systems, such as exosomes or viral vectors,
capable of crossing the BBB and delivering
therapeutics to specific cell types within the brain.
• 	 Intracerebroventricular administration: Directly
administering therapeutics into the cerebrospinal
fluid (CSF) via intracerebroventricular injection,
bypassing the BBB and ensuring widespread
distribution within the brain.
These approaches hold promise for improving drug delivery to the inflamed brain in TBI and enhancing the efficacy of neuroinflammation and neurorepair-targetedDooley et al. Journal of Neuroinflammation(2024) 21:183

therapies. Here we focus specifically on the potential
for gene delivery approaches to treat TBI. Gene delivery
employs specialized vectors and techniques enable thetransport therapeutic genetic material into target brain
cells (e.g., microglia, astrocytes), facilitating local production of therapeutic proteins (Fig. 1). Following entry
into the cells, the nucleic acid, either delivered directly as
an mRNA or delivered as a DNA gene and transcribed
into mRNA, is translated into the therapeutic protein
desired. While relying on the same technological tool kit
as gene therapy, gene delivery is conceptually distinct.
Gene therapy targets cells with a defective gene, relying on genetic compensation or correction to benefit
the targeted cell in cis. Examples of gene therapy consistof gene replacement or insertion of a corrected gene, inthe case of repairing loss-of-function mutations, or theinactivation of a malfunctioning gene, in the case of gainof-function mutations [47]. Gene delivery is a related
therapeutic concept, using the genetic cargo to produce
a modulating protein, with the capacity to improve tissue
physiology through secreted proteins or altered cell-cellinteractions. In effect, gene delivery uses healthy targeted

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cells as a “production factory” for a therapeutic payload,
acting in trans on third-party cells (Fig. 2). With the biologic being locally produced, even proteins with very
short half-lives (e.g., native interleukin 2 lasts only minutes) can become bioavailable at a sustained plateau level,
without the need for multiple invasive readministrations.
Advantages of gene delivery over gene therapy include
the capacity to target a diverse set of third-party cells fordelivery, and the ability of therapy amplification throughthe use of secreted beneficial cargos capable of aiding
multiple affected cells. While gene delivery therapeutics
face unique barriers of entry to the clinic, as an advanced
therapeutic, the ability to produce therapeutic proteins
within the brain potentially overcomes a key limitation ofsmall molecules and systemic delivery of biologics.
Modalities for neuro gene delivery

There are several different types of gene delivery systems,of which the main division is between non-viral vector gene delivery systems and viral vector gene delivery
systems (Fig. 2). Non-viral gene delivery systems introduce genetic materials into a host without the use of a

Fig. 1 Gene therapy versus gene delivery. Gene therapy (top) and gene delivery (bottom) are based on the same technological approaches of nucleic
acid delivery, however have conceptual differences in implementation. During gene therapy, the aim is to replace or repair a mutant gene with a functional replacement, in a cis-acting manner that improves the function of edited dysfunctional cells. During gene delivery, by contrast, the gene delivery
target is healthy, functional cells, which then produce and secrete the gene delivered cargo to improve the functionality in trans of cells in the local
environment

Dooley et al. Journal of Neuroinflammation(2024) 21:183

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Fig. 2 Modalities of gene delivery. Multiple approaches can be taken for gene delivery, including naked nucleic acids, encapsulated nucleic acids orvector-encoded nucleic acids, and using either DNA or RNA as the nucleic acid. Dependent on the delivery system is the route availability, with vectorencoded delivery systems capable of crossing the blood-brain barrier. Regardless of the original modality, the gene delivery harnesses a local cell to act
as the factory for protein production. Astrocytes are a key target cell type for the “factory cell”, being abundant, robust and specialised for secretion, while
microglia also have potential for utilisation. For the cargo to become effective, the proteins are then secreted to impact neighbouring cells in the local
environment. In TBI, several distinct pathways may be efficacious targets of these biologic cargos

virus. These non-viral delivery approaches include both
physical and chemical methods [48]. Physical methodsof non-viral gene delivery include use of naked DNA or
mRNA via injections (needle injection and ballistic DNA
injection) and poration-based methods (sono-poration,
photo-poration, magneto-fection, and hydro-poration).
Chemical methods encapsulate the DNA or mRNA cargowith either polymers or liposomes, which can then be
taken up through the process of endocytosis. In bothcases, the transfected cells become capable of producing the encoded protein cargo, through transcription
and translation (with DNA cargos) or direct translation(mRNA cargos). In the context of TBI, non-viral vectors
would rely on direct injection of the delivery cargo into
the brain tissue, as peripheral delivery does not cross the
blood-brain barrier and allow cargo production in thebrain.
Viral vector gene delivery systems, by contrast, exploit
the function of viruses to insert genetic material insidea host cell. As with non-viral vectors, viral vector genedelivery systems can carry DNA or RNA-based cargos. DNA-based viral vectors have the advantages ofbeing longer lasting, potentially indefinite for those that

integrate into the host genome, providing long-lastingcargo production. Examples of DNA-based viral vectors
include herpes virus, human foamy virus, bacteriophage,
poxvirus, lentivirus, adenovirus, and adeno-associatedvirus (AAV). Unlike DNA-based viral vectors, RNAbased viral vector delivery systems tend to be more shortlived, although RNA-based viral vectors have the benefitof direct translation of the RNA transcript into protein
cargoes, allowing for a rapid delivery system [49]. Eachviral vector gene delivery system has potential advantages and limitations to consider.
Relative suitability of viral vectors for neuro-delivery

In the context of TBI, selecting an appropriate viral vector for gene delivery hinges on distinct considerations.
The ideal vector for gene delivery TBI treatment should
demonstrate specific tropism for neuronal tissues, ensuring targeted transduction while minimizing off-target
effects. Moreover, the vector should sustain therapeutic cargo expression at optimal levels and durations
tailored to TBI pathology. Inadequate expression may
yield suboptimal therapeutic outcomes, while excessive
expression levels could trigger cytotoxicity or immune

Dooley et al. Journal of Neuroinflammation(2024) 21:183

responses. Mitigating vector-related pathologies andimmune responses is critical. Certain viral vectors may
induce cell lysis or elicit strong immune reactions, posing risks during vector production or patient administration. Scalable manufacturing processes adhering totherapeutic guidelines are indispensable for practicalapplication in TBI treatment, ensuring sufficient vector
production while meeting safety standards. Addressingthese criteria requires optimizing viral vector selection
for gene delivery in TBI, enhancing therapeutic efficacy
while minimizing adverse effects. Recent advancements
in gene delivery research have expanded the repertoireof potential viral vectors for treating TBI. Potential vectors include retrovirus, lentivirus, herpes virus, bacteriophage, adenovirus vectors (AdV), and adeno-associatedviral vectors (AAV). Each vector offers unique advantages
and challenges, underscoring the importance of evaluating their suitability for targeted gene delivery approaches
in TBI treatment. One of the most important attributes
is the vector trophism, with those vectors recognising
receptors on the brain vasculature often being capable ofnatural blood-brain barrier crossing.
Retroviruses, modified into the first viral vectors inclinical trials for in vivo gene therapy, are enveloped
spherical virus containing RNA as their genetic material. Retrovirus vectors possess the unique ability toreverse transcribe their single-stranded RNA into double-stranded DNA, subsequently integrating it into thehost cell’s genome [50]. Retroviruses can be categorized
as simple or complex. Simple retrovirus (e.g. Moloneymurine leukemia virus; gammaretrovirus) contains thegag gene (viral core), pol gene (reverse transcriptase andintegrase), and env gene (surface and transmembrane elements of viral envelope proteins). Complex retrovirus(e.g. HIV-1, lentivirus) contains all three genes of a simple retrovirus with the addition of other proteins. Vectors
derived from simple retroviruses (e.g. Moloney murine
leukemia virus) require the host cell to undergo division
for transduction. In contrast, vectors derived from complex retroviruses (e.g. HIV-1, lentivirus) can transduce
non-dividing cells [51]. While simple retroviruses havebeen employed to study aspects of neurogenesis, lentiviral vectors have become the predominant retroviral
vector for gene delivery to the CNS. While the lentiviral
vector continues to be used in research, and several vectors are progressing into the clinical trials space for neurological targets (Table 1), the safety of the vector is still
unclear. The key concern of these vectors lies in the riskof insertional mutagenesis, where integration into thehost genome could disrupt gene expression and lead toadverse effects. While integration was initially perceived
as advantageous for stable transgene expression, its
associated risks necessitate safer vector designs for genetherapy applications. To address this, modern vectors are

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engineered to minimize or eliminate integration, reducing the risk of insertional mutagenesis. Innovations in
vector genetics have yielded self-inactivating and nonintegrating lentiviral vectors, reducing integration risk
and vector-related pathologies. Nonintegrating lentiviral
vectors, at the forefront of retroviral vector development,show potential for CNS gene delivery. However, a limitation of nonintegrating lentiviral vectors lies in poorly
characterized transgene expression duration and level.
While studies demonstrate transgene persistence up to
3 months in the rat brain and up to 9 months in othertissues, extensive research is needed to ascertain expression limits [52, 53]. Addressing this shortfall is crucial for
optimizing gene therapy efficacy and safety in CNS applications, ensuring adequate and sustainable transgene
expression while minimizing the risk of adverse effects.
Herpes simplex virus 1 (HSV-1) is an oncolytic virus
with a double-stranded DNA genome and can be transmitted through contact with an infected person’s lesions,mucosal surfaces, genital secretions, or oral secretions.
The brain-trophic capacity of the virus [54] allows engineered forms to be used therapeutically. Oncolytic versions of HSV-1 have been used as immunovirotherapies
for multiple cancers, including in the context of glioblastoma [55–58]. While these viral vectors can be engineered to include a therapeutic payload [59], the current
focus as an oncolytic vector make them unsuitable foruse in TBI gene delivery at this stage.
Bacteriophage are a novel class of virus for gene delivery [60]. Repurposed from the natural infection host ofbacteria, humans have little-to-no pre-existing immunity
with good tolerance in clinical trials [61]. Filamentous,
rod-shaped phages such as f1, fd, and most importantly
M13 are widely used in a variety of biochemical andbiomedical applications. While not naturally capable of
BBB-crossing, modification through phage display has
allowed successful penetration using the Trojan horsemechanism [62, 63]. Hybrid vectors can also be generated, combining aspects of bacteriophages and othervectors [64]. Biopanning has even identified novel spatiotemporal targeting motifs enhancing BBB-crossing during TBI [65]. Bacteriophage vectors are still under early
development, however, with further research needed tounderstand the potential and limitations of phage-based
therapies for neurological delivery.
Adenovirus (AdV) is a non-enveloped virus with adouble-stranded DNA genome. AdV vectors have beenused in gene therapy to treat macular degeneration, cystic fibrosis, and several types of cancers [66]. AdV haveseveral advantages in comparison to retroviral vectors,
with a wide range of cell trophism, a lower risk of chromosome mutagenesis, and low rates of viral gene integration of the host’s genome [67]. Some noted challenges
with using AdV include off-target cellular inclusion due

Dooley et al. Journal of Neuroinflammation(2024) 21:183

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Table 1 Clinical trials of viral vectors for gene delivery or gene therapy in the brain. Data collated from www.clinicaltrials.gov., basedon “gene delivery” or “gene therapy” combined with “brain” or “central nervous system”. The list was supplemented through additional
searches using “AAV”, “lentivirus”, “retrovirus” combined with “neuroinflammation”, “neurodegeneration”. Output was manually curated forthe use of viral vectors in the brain
ClinicalTrials.gov Identifier
NCT04747431
NCT02418598, NCT01973543, NCT03562494,
NCT00229736, NCT03065192
NCT05040217
NCT00151216
NCT05894343, NCT05603312, NCT00643890,
NCT00195143
NCT04680065
NCT06285643, NCT01621581, NCT04167540
NCT00876863
NCT00087789
NCT00252850, NCT00985517
NCT05243017, NCT04120493
NCT03300453
NCT05394064
NCT05518188
NCT05317780
NCT04998396
NCT04884815
NCT03770572
NCT05228145
NCT04273243, NCT02725580
NCT04737460
NCT05419492, NCT06112275, NCT06283212
NCT04127578
NCT04411654
NCT03952637
NCT04798235
NCT03315182
NCT02716246, NCT04088734
NCT04571970, NCT03566043
NCT03580083, NCT02702115
NCT05152823
NCT02362438
NCT05606614, NCT06152237
NCT04408625, NCT06064890
NCT05335876, NCT03461289, NCT03306277,
NCT03381729, NCT03505099, NCT03837184,
NCT04042025, NCT05073133, NCT04851873,
NCT05386680, NCT05089656, NCT05901987,
NCT05824169, NCT02122952, NCT06288230
NCT06063850
NCT04133454
NCT04771416
NCT04713475
NCT05400330, NCT03634007

Conditions
Frontotemporal Dementia
Parkinson’s Disease

Vector and cargo
AAV1.GRN
AAV2.AADC

RouteIntrathecal
Intraparenchymal

Alzheimer’s Disease
Batten Disease
Parkinson’s Disease

AAV2.BDNF
AAV2.CLN2
AAV2.GAD

Intraparenchymal
Intraparenchymal
Intraparenchymal

Multiple System Atrophy
Parkinson’s Disease
Alzheimer’s Disease
Alzheimer’s Disease
Parkinson’s Disease
Huntington Disease
Mucopolysaccharidosis IIIB (Sanfilippo
Syndrome)
Adrenomyeloneuropathy
Spastic Paraplegia Type 50
Canavan disease

AAV2.GDNF
AAV2.GDNF
AAV2.NGF
AAV2.NGF
AAV2.NTN
AAV5.miR-HTT
AAV5.NAGLU

Intraparenchymal
Intraparenchymal
Intraparenchymal
Intraparenchymal
Intraparenchymal
Intraparenchymal
Intraparenchymal

AAV9.ABCD1
AAV9.AP4M1
AAV9.ASPA

Canavan Disease
Wilson Disease
Batten Disease
Batten Disease
Batten Disease
Batten disease
Dravet Syndrome
Parkinson’s Disease
Type 2 Gaucher Disease
Tay-Sachs Disease, Sandhoff Disease
Tay-Sachs Disease, Sandhoff Disease
Mucopolysaccharidosis IIIB (Sanfilippo
Syndrome)
Mucopolysaccharidosis IIIA (Sanfilippo Syndrome)
Mucopolysaccharidosis II (Hunter
Syndrome)
Mucopolysaccharidosis I
IGHMBP2-Related Diseases
Giant Axonal Neuropathy
Rett Syndrome
Frontotemporal Dementia
Spinal Muscular Atrophy

AAV9.ASPA
AAV9.ATP7B
AAV9.CLN3
AAV9.CLN5
AAV9.CLN6
AAV9.CLN7
AAV9.eTF-SCN1A
AAV9.GBA
AAV9.GBA1
AAV9.GLB1
AAV9.HEXA/HEXB
AAV9.hNAGLU

Intrathecal
Intrathecal
Intravenous,
intraparenchymal
Intravenous
Intravenous
Intrathecal
Intraparenchymal
Intrathecal
Intrathecal
Intraparenchymal
Intraparenchymal
Intrathecal
Intravenous
Intrathecal
Intravenous

AAV9.hSGSH

Intravenous

AAV9.IDS

Intrathecal,
intraparenchymal
Intravenous, intrathecal
Intrathecal
Intrathecal
Intrathecal
Intraparenchymal
Intravenous, intrathecal

Refractory Epilepsy
Alzheimer’s Disease
Early Infantile Krabbe Disease
GM1 Gangliosidosis
Alzheimer’s Disease

AAV9.Syn1-miR-GRIK2
AAV9.TERTAAVhu68.GALCAAVhu68.GLB1
AAVrh10.APOE2

AAV9.IDUA
AAV9.IGHMBP2
AAV9.JeT-GAN
AAV9.MECP2
AAV9.PGRN
AAV9.SMN1

Intraparenchymal
Intravenous, intrathecal
Intrathecal
Intrathecal
Intraparenchymal,
intrathecal

Dooley et al. Journal of Neuroinflammation(2024) 21:183

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Table 1 (continued)
ClinicalTrials.gov Identifier
NCT01801709

Vector and cargo
AAVrh10.ARSA

RouteIntraparenchymal

AAVrh10.CLN2
AAVrh10.CYP46A1
AAVrh10.GALCAAVrh10.GLB1
AAVrh10.hSGSH

Intraparenchymal
Intraparenchymal
Intravenous
Intrathecal
Intraparenchymal

NCT06100276
NCT04669535

Conditions
Early Onset Metachromatic
Leukodystrophy
Batten Disease
Huntington Disease
Krabbe Disease
GM1 Gangliosidosis
Mucopolysaccharidosis IIIA (Sanfilippo Syndrome)
Amyotrophic Lateral Sclerosis
Tay-Sachs Disease, Sandhoff Disease

AAVrh10.miR-SOD1
AAVrh8.HEXA/HEXB

NCT00002824, NCT00870181, NCT00589875

Glioblastoma

AdV.HSV-TK

NCT00031083
NCT05686798

Glioblastoma
Glioblastoma

AdV.IFNβ
AdV.mutTKSR39rep

NCT00004041, NCT00004080
NCT04601974
NCT03720418, NCT00627588, NCT01856439
NCT03727555
NCT04833907
NCT01470794, NCT01156584, NCT04327011,
NCT01985256, NCT02414165

Glioblastoma
Refractory Epilepsy
Parkinson’s Disease
X-linked Adrenoleukodystrophy
Canavan Disease
Glioblastoma

AdV.p53
LV.EKC
LV.TH.AADC.CH1
LV.TYF-ABCD1
rAAV-Olig001.ASPA
Retroviral vectors
Toca511 and TocaFC

Intrathecal
Intraparenchymal,
intrathecal
Intratumoral, resection
cavityIntratumoral
Intratumoral, resection
cavityIntratumoral
Intraparenchymal
Intraparenchymal
Intraparenchymal
Intraparenchymal
Intravenous, intratumoral, intrathecal

NCT01414985, NCT01161576
NCT05541627
NCT04693598, NCT05739643
NCT04273269
NCT03612869

to nonspecificity, immunodominance, and re-gained
replication proficiency [66]. Due to these issues, in thebrain context, AdV vectors have largely entered clinical
trials for use in glioblastoma, where they have delivered
various anti-oncogenic or pro-inflammatory payloads[68–70]. Further development of the AdV vector systemmay, however, yield a vector appropriate for use in the
TBI context.
The most frequently used viral vectors for the brain are
the Adenovirus Associated Virus (AAV)-derived vectors.
AAV are non-enveloped virus with a single-stranded
DNA genome, and are widely used as viral vectors due tothe diversity of stereotypes, with varying displays of tissue tropism, and relatively low antigenicity [71]. Targeting the brain with AAV vectors requires selecting AAV
serotypes with tropism for neural tissues, coupled withappropriate delivery systems. 13 naturally occurring
AAV serotypes have been identified, among which many
exhibit varying degrees of efficiency in infecting brain
cells upon direct delivery. However, among the natural
serotypes, only AAV9 and the less-explored AAVrh.10
have been found to possess the ability to cross the BBB[72, 73], while the other serotypes require direct delivery.
Finally, there is a rich field of AAV improvements which
includes modification of the ancestral AAV vectors foraltered trophism [74]. Several modifications of AAV9, inparticular, have useful brain trophisms. While the initial
generation of the PHPb capsid for enhanced blood-brain
barrier crossing is limited in clinical utility due to its

use of a mouse-specific endothelial binding [75], similar
approaches in non-human primates have generated vectors with improved efficacy of delivery to the primate
brain [76], and the area is active in development.
The package size of AAVs, the maximum genetic material they can carry, is relatively modest at around 4.7–
5.2 kb, limiting the size of therapeutic genes deliverable[77]. The broad trophism of many AAV serotypes canalso result in adverse effects from off-target transduction,such as acute liver damage with AAV9 [78]. Despite thesechallenges AAV-based therapies hold promise for genetic
treatment of diseases affecting the brain. Indeed, of theviral vectors currently being trialled in the neurologicalspace, the majority use an AAV-based system (Table 1),
with multiple ongoing clinical trials to treat neurogenerative disorders such as Parkinson’s disease, Alzheimer’s
disease, and Amyotrophic Lateral Sclerosis. To date, prototype vectors based on natural seroptypes AAV1, AAV2,
AAV5, AAV9, AAVhu86, AAVrh10 and AAVrh8, andthe novel capsid AAV.Oligo001, have been employed inclinical applications targeting the central nervous system.
Among these, AAV9 stands out as the most frequently
utilized serotype for CNS gene therapy applications [79,80] (Fig. 3A).
Delivery routes to the brain

The main routes of vector introduction for neuro-delivery are intraparenchymal administration, intravenous
administration, intra-CSF administration and intranasal

Dooley et al. Journal of Neuroinflammation(2024) 21:183

Page 9 of 15

Fig. 3 Relative use of viral vectors and delivery routes for gene therapy or gene delivery to the brain. Distribution of current and past clinical trials registered on ClinicalTrials.gov for brain viral vectors (listed in Table 1), based on the number (upper left) of unique products. (A) Relative use of viral vector,and (B) delivery routeadministration (Fig. 3B). Each administration route has
its set of advantages and disadvantages.
Intravenous Delivery. Intravenous method allows for
systemic gene delivery, providing widespread accessto the brain without the need for invasive procedures.
Large molecules, including most gene delivery vectors,
are often unable to penetrate the BBB efficiently, limiting their effectiveness in treating neurological disorders. Several AAVs, most notable AAV9 [81], overcome
this by utilizing natural tropism for the central nervous
system and the ability to bypass the BBB. The approvalof AAV9.SMN1, with intravenous delivery of AAV9 for
SMN1 gene therapy to lower motor neurons, marked significant advancement in gene therapy for treating neurological disorders [82]. While AAV9 remains the primary
choice in clinical trials, other serotypes such as rAAV8,rAAVrh.10, rAAVrh.39, rAAVrh.43, and rAAV7 have also
been utilized for BBB-crossing in pre-clinical models[83]. The key disadvantage of intravenous delivery is thelimited efficiency in crossing the BBB, which can resultin lower transduction rates, reduced therapeutic efficacy
and off-target effects compared to direct CNS injection
methods. The use of tissue-specific promoters in AAV

therapeutics are potentially viable routes to mitigate
some of these issues [84, 85].
Intraparenchymal delivery. For vectors with brain trophism but poor BBB crossing, such as AAV2, direct
delivery approaches are required. The intraparenchymal
injection method for gene delivery involves direct targeting of the site of pathology. This approach is highly effective for diseases affecting specific anatomical regions of
the brain. In Canavan Disease, which is most evident insubcortical white matter, larger brain areas were targetedthrough twelve injection sites in the first-ever intraparenchymal clinical trial utilizing AAV [86]. Many viral
vectors exhibit limited diffusion capabilities, which creates a trade-off between highly targeted delivery andpoor distribution when using intraparenchymal delivery. A further trade-off is created with minimizing systemic exposure, reducing the risk of off-target effects
and enhancing localized effects, but requiring an invasive
surgical procedure which inherently carries risks. These
drawbacks underscore the importance of careful consideration and optimization of delivery methods in AAV
gene therapy approaches.

Dooley et al. Journal of Neuroinflammation(2024) 21:183

CSF delivery. Intracerebroventricular, intracisternal
magna, and intrathecal injections are utilized to delivervectors via the CSF, facilitating widespread distribution
throughout the central nervous system. While each provides direct exposure to the CSF, the route of CSF entry
may influence the distribution and efficacy of gene delivery systems [87, 88]. Intracerebroventricular injections ofrecombinant adeno-associated virus (e.g., AAV2, AAV4,
AAV5 and AAV9) have been observed to transduce primary ependymal cells specifically in the choroid plexus
but not in other regions of the brain in adult mice andrats. Delivery of vectors into the CSF can also be accomplished intracisternal magna injection for AAV, although
this method is not widely utilized in clinical settings.
Recent animal studies indicate that intracisternal magna
and intrathecal vector delivery are both safe and effective for AAV (e.g., AAV2, AAV4, AAV5 and AAV9) genedelivery, with intracisternal magna potentially targetingthe brain more extensively than intrathecal delivery [89–91]. CSF delivery moderates both the risks and advantages of intravenous versus intraparenchymal delivery,
with moderate invasiveness and intermediate off-target
dosage issues. CSF delivery provides for wider distribution across the brain than intraparenchymal injection,
however alterations in CSF flow dynamics can provide
variable spatial distribution.
Intranasal delivery. Finally, intranasal delivery provides
a non-invasive strategy for directing therapeutic agentsfrom the nasal cavity to the brain, bypassing the BBB
and mitigating systemic exposure [92]. While intranasal delivery in principle offers the numerous advantages,
it faces limitations such as low delivery efficiency andvariable and unpredictable distribution of therapeutic
agents within the brain. Additionally, the relatively small
fraction of administered drugs that reaches the brain
necessitates optimization strategies to enhance delivery
efficiency and achieve precise targeting [93, 94]. FUSmediated intranasal delivery (FUSIN) of AAV represents
a promising advance addressing the limitations of conventional intranasal delivery methods. Notably, FUSIN
has demonstrated success in targeting both superficial
regions like the cortex, midbrain, pons and deeper brain
structures such as the brainstem [95]. Despite the current
lack of clinical testing, the attractiveness of this approachlies in its potential to combine a non-invasive approachwith low doses and fewer off-target effects.
Potential therapeutic cargos for TBI treatment

With injury kinetics amenable to intervention, and viable
vectors and administration routes for gene delivery, there
is a plethora of potential therapeutic cargos that could beincorporated into TBI gene delivery. While a comprehensive analysis of potential therapeutic cargos is not possible, the growing understanding of the pathophysiology ofPage 10 of 15

neurodysregulation in the post-TBI brain suggests representative cargos across several independent and interdependent pathways, in particular inflammation, glutamate
excitotoxicity, and oxidative stress [96].
Neuroinflammation represents a powerful driver of
secondary injury with many potentially modifiable targets acting through soluble mediators suitable for genedelivery systems. The deleterious effects of excessive
inflammatory mediators such as interleukin 1β (IL1β) andtumour necrosis factor (TNF) on neurons following TBI
are multifactorial, leading to neuronal death through synergistic pathways [97, 98]. Directly inhibiting the effects
of inflammatory mediators by upregulating expressionof their receptors’ natural antagonists (e.g. IL-1ra) would
negate these deleterious effects; systemic administrationof recombinant IL-1ra has been shown to modulate brain
cytokine concentrations in severe human TBI [99], butwould inevitably act on multiple organ systems rather
than solely in the brain. Alternatively, enhancing production of predominantly anti-inflammatory cytokines suchas interleukin 10 (IL-10). IL-10 has pleiotropic effects
across the inflammatory cascade, including protection
against neurotoxic microglial hyperactivation [100], andhas been effective in neurodegeneration models using
gene delivery [101]. IL-6, while often acting as an inflammatory cytokine in the periphery, may have anti-inflammatory properties in the brain during TBI, as it drives
microglia to a restorative phenotype in pre-clinical models [43, 102]. The migration of circulating inflammatory
cells could be impeded through the production of dominant negative chemokines, as demonstrated throughAAV-gene delivery in Alzheimer’s Disease mice [103].
Lastly, cellular regulators of the inflammatory responsesuch as regulatory T cells are present in the brain, where
they have potent anti-inflammatory properties as well
as exerting neuroprotective and neurorepair programs[104]. This cellular pathway can be amplified throughrecruitment to areas of brain injury, such as through thelocal production of chemokines [105], or through thelocal production of interleukin 2 (IL-2) to maintain boththeir survival and function [29]. The latter approach iseffective when delivered using intravenous AAV-based
expression in pre-clinical models, providing proof-ofprinciple of the gene delivery approach.
Glutamate excitotoxicity is thought to occur from an
inability of excitatory amino acid transporters (EAATs)
to move excess synaptic glutamate intracellularly [106–108]. This leads to hyperactivation of AMPA and NMDA
receptors, with resultant detrimental elevations in intracellular calcium and reactive oxygen species release,leading to neuronal death [96]. Upregulating EAAT
expression directly (e.g. via SLC1A2) has the potential toredress the balance between extracellular and intracellular glutamate [109], however to function in a trans-acting

Dooley et al. Journal of Neuroinflammation(2024) 21:183

gene delivery manner, the optimal approach may be todeliver secreted factors that protect neurons against the
detrimental effects of excess glutamate, such as insulin[110] or BDNF [111]. AAV-mediated delivery of BDNF
was indeed demonstrated to be effective in a pre-clinicalmodel of Alzheimer’s Disease, providing proof-of-principle of the approach.
Oxidative stress, the result of an imbalance between
reactive oxygen species production and compensatoryanti-oxidant mechanisms, occurs via a number of contemporaneous processes in TBI including mitochondrial
dysfunction, inflammation and ischaemia-reperfusion[112, 113]. Oxidative stress causes direct neural injury
as well as exacerbating mitochondrial dysfunction andcompromising blood-brain barrier integrity [112]. In
principle the cellular impact of oxidative stress can be
reduced by upregulating local production of extracellular
regulators of the redox environment, such as superoxide
dismutase, catalases, thioltransferases or peroxiredoxins[114]. Production and secretion of these cargos, throughgene delivery, may dampen the oxidative stress of neurons within the damaged brain.
Finally, gene delivery cargos are not limited to endogenous proteins. Xenoproteins with appropriate properties
can be co-opted for beneficial effects in TBI, assuming
immunogenicity issues are not present. As an exampleof this approach is the chondroitinase ABC (ChABC)
enzyme from Proteus vulgaris. ChABC synergies withseveral of the pathways above, increasing IL-10 for its
anti-inflammatory properties [115–117] and digesting
the chondroitin sulfate proteoglycans that accumulate
after neuroinjury and impede repair in rodent neurotrauma models [118]. Direct delivery of the enzyme tothe central nervous system of rats is beneficial after spinal
cord injury [118] and may improve outcomes after TBI[119–121]. Testing of these and other candidate xenoproteins via gene delivery in pre-clinical models of TBI may
identify lead cargos to move into clinical trials.
Limitations of gene delivery

While gene delivery systems have key advantages in thepotential use as TBI therapeutics, there are also important limitations to be considered. Gene delivery systems
are limited to the delivery of DNA-encoded biologics,
usually proteins, and are unsuitable for small moleculedelivery. Cargo expression is relatively slow, creating
biokinetics which are unfavourable to rapid pathological events. Gene expression that is wide-spread has
high potential for adverse events in off-target tissues,
depending on the biologic encoded. The proteins capable of being encoded, and the regulatory features of theencoded, are also limited by the cargo size, which, in thecase of AAV-based systems, is restrictive [77]. Vectors
can have toxicity issues, especially at high doses [122].

Page 11 of 15

The efficiency of natural vectors can be impeded by priorexposure, while even engineered vectors can be blocked
by immune responses upon re-administration. The useof engineered vectors with sustained production alleviates the need for multiple exposure, however this gives
rise to the potential problem of production outlasting
the desired intervention period. Many of these problems
can be engineered around, through either capsid or cargo
design. For example, cargo production can be restricted
to particular tissues through the use of cell type-specificpromoters, and temporal control can be created throughlinking cargo expression to small molecule-inducers
with a short half-life [29]. Nonetheless, the engineering
challenges are formidable. The design of the therapeutic agents will also need to take into careful account theheterogeneity of patients, treatment windows, suitability
for long-term administration, and the risk profile of vectors and routes. Beyond the technical challenges, genedelivery treatments face a different regulatory frameworkand higher economic barriers of entry compared to small
molecule treatments.
The health economics of gene delivery in TBI

A frequent challenge for advanced therapeutics is the
economic viability, raising the issue of whether the healtheconomic landscape of TBI can support the developmentof advanced therapeutics. The economic consequences of
TBI are vast, with an estimated total cost worldwide of
US$400 billion, or 0.5% of the entire annual global output [2]. In more tangible terms, per-patient in-hospitaland post-discharge care costs ~$88,000 and >$2 million
respectively following severe TBI [123, 124], before evenconsidering the subsequent loss of the individuals’ economic productivity. Judging the cost-effectiveness oftreatments for acute TBI is complex, as the economic
consequences of TBI are multifaceted, and are in many
ways inverted in comparison to the majority of medical
conditions. In most diseases, the natural history is one ofprogressive debility over time, and treatments are judged
on their ability to delay this accrual of damage; the inherent health and care costs are often delayed rather thanprevented (for example in the context of disseminatedmalignancy), and therefore the cost-effectiveness of treatments is judged against the number of “quality-adjusted
life years” (QALYs) afforded by the treatment. Whilst
QALYs are certainly an important consideration in theeconomic evaluation of TBI, the substantial health andcare costs that are currently necessarily generated, andfront-loaded, by TBI mean that effective acute treatments
stand to create substantial savings in costs before theconcept of cost-per-QALY even becomes relevant. The
median length of ITU admission for patients with severe
TBI is 21 days [44] at a cost of $1900 per day [125], followed by an average rehabilitation cost of £50,000 perDooley et al. Journal of Neuroinflammation(2024) 21:183

patient prior to discharge back to the community [126],
i.e. a total inpatient cost of approximately £$100,000 per
patient. Following discharge from the hospital, patientsrequire varying levels of care and support for activities
of daily living, with the estimated lifetime post-dischargecare costs averaging $2.5 million per patient [124].
In addition to the direct health-care costs, the average loss in QALY needs to be assessed. TBI are skewed
towards a young demographic, with a median age of
injury between 38 and 50 [3], which compounds thelong-term cost of disability and care and amplifies theloss of economic productivity. Using the QALY calculations for the cost-effectiveness study of the CRASH-3
trial, the average UK patient with mild to moderate TBI
lost 4.77 QALY over their average 16.87 life years postTBI [127]. The cost-per-QALY used varies across healthcare systems, however even using a relatively low value,such as used in the United Kingdom of ~$40,000 per
QALY, the aggregate direct healthcare costs and QALY
costs average at $2.5 million per TBI patient. This allows
for even relatively modest reductions in patient costs or
improvements in patient quality of life are economically
viable. For example, the RAIN study (n = 2665) assessing
the simple intervention of early transfer of TBI patientsto a dedicated neurocritical care unit, resulted in an additional 1.8 QALYs per patient, valued at ~$70,000 under
the local QALY valuation [128]. Indeed, it is estimatedthat the simple implementation of the Brain Trauma
Foundation Clinical Guidelines (a consensus statement
stipulating standards of care in TBI) leads to a total
reduction in healthcare costs of $175,479 per patientwith severe TBI [129].
This health economics analysis under-estimates thesocietal benefits of improved treatments for TBI. The“hidden disability” of cognitive impact can lead to isolation, economic hardship, and impacts on both mental
health and relationships. It is estimated that the loss of
economic productivity accounts for an even greater proportion of the total economic impact of TBI than the
direct healthcare costs [130]. Families of patients with
TBI are often unequipped for the financial, emotional,
and logistical burdens of caring for patients with longterm disability. Brain injuries can cause hormonal, behavioral, and emotional changes, with a profound impact on
relationships; divorce rates are significantly higher following TBI [131], and long-term aggressive behaviors are
frequent [132]. Family members often assume the role ofcarer at the detriment to their careers and livelihoods.
These effects can be conservatively estimated at a totalsocietal cost of $4 million per patient [133].
Given the complexity and substantial reach of the economic consequences of TBI, even highly expensive therapies given early in the disease course have the potential
to result in substantial economic benefits overall if they

Page 12 of 15

successfully improve long-term outcomes. However, as
TBI ranges in severity from trivial through to immediately life-limiting injuries, identifying patient groups who
are most likely to benefit will be essential. Judging thecost-effectiveness of treatments such as AAV gene therapy in this setting would necessitate the involvement ofhealth economists with a strong grounding in TBI.

Conclusions
Gene delivery is a promising avenue for therapeutic
treatment in the chronic stages of TBI. Gene delivery
approaches have the benefit of allowing sustained production of biologics within the affected tissue, bypassing
the intrinsic limitations present when using many small
molecules or biologics in the brain. While gene delivery
is not currently used in TBI, the vector development programs implemented for diverse neurological conditions
provide a fertile list of candidates for delivery approaches.
Currently, AAV9/AAVrh10-based intravenous delivery
or AAV2/AAV9-based intra-CSF delivery are the mostadvanced approaches for neurodelivery, however exciting developments in vector design and route delivery areon the horizon. Recent advances in understanding thepathophysiology of the chronic phase of TBI also highlight multiple attractive endogenous and xenoprotein
cargos, with the potential to dampen down neuroinflammation, or protect against glutamate excitotoxicity andoxidative stress. While the paucity of clinical researchof gene delivery in TBI is, in part, due to the recency of
the technology advances, advanced therapies have, in
general, made poor penetration into TBI clinical trials.
Relative to the number of patients, there are more than
100-fold fewer TBI clinical trials than are run for comparable neurological conditions such as multiple sclerosis
and glioblastoma. While issues such as translatability ofanimal models and heterogeneity of pathology are present in TBI, as in other indications, the success of surgical intervention clinical trials in improving survival andpost-TBI outcomes argues against a general capacity to
run a clinical trial in TBI. The advantages of gene delivery in TBI, in particular the compatibility of pharmacodynamics to injury kinetics, and the viability of the healtheconomics, suggest gene delivery therapeutics may be a
productive avenue of research.
Acknowledgements
not applicable.
Author contributions
All authors drafted and approved the manuscript.
Funding
This work was supported by the MRC DPFS grant MR/X029166/1.
Data availability
No datasets were generated or analysed during the current study.

Dooley et al. Journal of Neuroinflammation(2024) 21:183

Declarations
Ethical approval
Not applicable.
Consent for publication
Not applicable.
Competing interests
J.D. and A.L. are coinventors on the patent application PCT/GB2020/052148
and are cofounders of Aila Biotech Ltd. E.J.N., J.D., and A.L. are potential
financial beneficiaries of Aila Biotech Ltd.
Received: 1 April 2024 / Accepted: 17 June 2024

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