Module 7

AAV & Lentiviral Gene Therapy

Viral-vector gene therapy delivers a functional copy of a gene to compensate for a loss-of-function mutation. AAVs dominate in vivo use (Luxturna, Zolgensma, Hemgenix, Elevidys). Lentiviruses integrate stably into the genome and are used ex vivo for hematopoietic or T-cell gene therapy.

1. AAV Biology & Serotypes

Adeno-associated virus is a small (~26 nm) non-enveloped ssDNA parvovirus with a 4.7 kb genome. Recombinant AAV vectors replace rep and capwith the therapeutic transgene; capsid serotype (AAV1–13 natural, plus engineered variants) determines tissue tropism. AAV9 crosses BBB in neonates; AAV8 targets liver; AAV-PHP.eB (Deverman 2016) is an engineered variant for neuronal targeting in mice.

2. Capsid Engineering

Directed evolution of capsid libraries identifies variants with preferred tropism. AAV-DJ, AAV-retro, AAV-MaCPNS (Goertsen 2022) each extend the natural repertoire. Machine-learning-guided capsid design (Bryant 2021, Ogden 2019) is now a routine complement. Primary challenge: pre-existing neutralising antibodies exclude 30–70% of patients from AAV therapies, depending on serotype and geography.

Simulation: Serotype Tropism

Python

script.py35 lines

import numpy as np, matplotlib
matplotlib.use('Agg')
import matplotlib.pyplot as plt

# AAV serotype tropism heat-map (simplified)
serotypes = ['AAV2','AAV5','AAV8','AAV9','AAV-DJ','AAV-PHP.eB']
tissues = ['Liver','Muscle','Lung','Heart','CNS','Retina']

tropism = np.array([
    [0.6, 0.3, 0.2, 0.3, 0.4, 0.9],
    [0.2, 0.2, 0.7, 0.2, 0.2, 0.4],
    [0.9, 0.5, 0.4, 0.7, 0.3, 0.3],
    [0.6, 0.7, 0.3, 0.9, 0.7, 0.2],
    [0.7, 0.5, 0.3, 0.6, 0.5, 0.3],
    [0.3, 0.3, 0.2, 0.3, 0.95, 0.2],
])

fig, ax = plt.subplots(figsize=(10, 6), facecolor='#0a0a1a')
ax.set_facecolor('#111827'); ax.tick_params(colors='#cbd5e1')
for s in ax.spines.values(): s.set_color('#334155')
im = ax.imshow(tropism, cmap='viridis', aspect='auto', vmin=0, vmax=1)
ax.set_xticks(range(len(tissues))); ax.set_xticklabels(tissues)
ax.set_yticks(range(len(serotypes))); ax.set_yticklabels(serotypes)
for i in range(len(serotypes)):
    for j in range(len(tissues)):
        ax.text(j, i, f'{tropism[i,j]:.1f}', ha='center',
                color='#fde68a' if tropism[i,j]<0.6 else '#0a0a1a',
                fontsize=9, fontweight='bold')
ax.set_title('AAV serotype tropism (relative)',
             color='#5eead4', fontweight='bold')
plt.colorbar(im, ax=ax)
plt.tight_layout()
plt.savefig('output.png', dpi=120, bbox_inches='tight', facecolor='#0a0a1a')
print('AAV-PHP.eB (Deverman 2016) crosses BBB via directed evolution')
print('AAV9 systemic delivery: Zolgensma ($2.1M SMA therapy)')

Click Run to execute the Python code

Code will be executed with Python 3 on the server

3. Approved AAV Therapies

Luxturna (voretigene, 2017): subretinal AAV2 delivering RPE65 for Leber congenital amaurosis; first FDA gene therapy for inherited disease.
Zolgensma (onasemnogene, 2019): IV AAV9 delivering SMN1 for spinal muscular atrophy; $2.1M per dose.
Hemgenix (etranacogene, 2022): AAV5 liver-directed factor IX for haemophilia B.
Elevidys (delandistrogene, 2023): AAVrh74 micro-dystrophin for Duchenne muscular dystrophy.
Roctavian (valoctocogene, 2023): factor VIII for haemophilia A.

4. Lentiviral Ex-Vivo Gene Therapy

Lentiviruses (HIV-derived, replication-incompetent) integrate stably into dividing and non-dividing cells. Ex-vivo lentiviral HSC gene therapy has been approved for ADA-SCID (Strimvelis), β-thalassemia (Zynteglo), cerebral ALD (Skysona), and metachromatic leukodystrophy (Libmeldy). Insertional mutagenesis was the historical concern (X-SCID trial leukemia 2000); modern self-inactivating vectors have drastically reduced risk.

Key References

• Russell, S. et al. (2017). “Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy.” Lancet, 390, 849–860.

• Mendell, J. R. et al. (2017). “Single-dose gene-replacement therapy for spinal muscular atrophy.” N. Engl. J. Med., 377, 1713–1722.

• Deverman, B. E. et al. (2016). “Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.” Nat. Biotechnol., 34, 204–209.

• Bryant, D. H. et al. (2021). “Deep diversification of an AAV capsid protein by machine learning.” Nat. Biotechnol., 39, 691–696.

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